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

BACKGROUND: 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.
MATERIALS AND METHODS: A total of 364 patients with type 2 diabetes and an estimated glomerular filtration rate (eGFR) ≥30 mL/min/1.73m2 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.
RESULTS: Seventy-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 (area under the curve 0.63, 95% confidence interval 0.57-0.70, p < 0.001), 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 (hazard ratio 2.27, 95% confidence interval 1.23-4.20, p = 0.009) and remained consistent across subgroups stratified by age, sex, systolic blood pressure, eGFR, albuminuria, and the use of renin-angiotensin system blockers.
CONCLUSIONS: Higher circulating levels of sTNFR2 are independently associated with an eGFR decline and progressive albuminuria in patients with type 2 diabetes.

Introduction

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 [1]. Approximately 35%-50% of patients with type 2 diabetes will eventually develop various kidney disorders, such as microalbuminuria, macroalbuminuria, or impaired renal function [1, 2]. Diabetic kidney disease is the most common cause of end stage renal disease (ESRD) worldwide [3], and the health care costs of DKD has increased substantially in the recent decades [2]. Chronic kidney disease is the dominant contributor to excess mortality in patients with type 2 diabetes [4]. To prevent ESRD and premature mortality, it is crucial to identify patients with high risk of advanced renal disease at an early stage and provide personalized management.

Screening of patients with diabetic nephropathy includes measuring their urinary albumin-creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR) at least once annually [5]. Although most patients with a risk of DKD can be identified by screening for albuminuria and eGFR, significant glomerular structural changes are often already developed by the time microalbuminuria becomes apparent [6]. In addition, patients with type 2 diabetes can develop renal impairments in the absence of albuminuria [7]; thus making albuminuria an ineffective screening test for these patients. Novel biomarkers are needed to detect the early stages of diabetic kidney disease.

Activation of the tumor necrosis factor-α pathway has emerged as an important mechanism in the pathogenesis of DKD [8]. Tumor necrosis factor-α is an inflammatory cytokine involved in a number of signaling pathways that lead to apoptosis and inflammatory processes, by interacting with two types of membrane receptors: tumor necrosis factor receptor (TNFR) 1 and TNFR2 [9]. The circulating level of soluble tumor necrosis factor receptor 2 (sTNFR2), which is released from the cell surface, is associated with end-stage renal disease, cardiovascular disease, and mortality in patients with type 2 diabetes [10-13]. However, the association of sTNFR2 with the development of early nephropathy in patients with type 2 diabetes has been investigated less often. Therefore, the aim of this study was to explore the association of sTNFR2 with early changes in diabetic nephropathy in patients with type 2 diabetes.

Materials and methods

Study design and participants

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/1.73m2, 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 (Quantikine ELISA kit; R&D Systems, Minneapolis, MN, USA). 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.

This 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.

Renal outcomes

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.

Statistical analyses

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 (IBM Corporation, Armonk, NY, USA).

Results

Study patients

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/m2. The baseline characteristics of the study population are summarized in Table 1.

Table 1

Baseline characteristics of the entire study population and according to the occurrence of the renal outcomes.

	Entire cohort	Occurrence of the renal outcomes		P value
	(n = 364)	Yes (n = 73)	No (n = 291)
Age	61.4±12.8	63.6±11.6	60.9±13.0	0.102
Male sex (%)	256 (70.3)	47 (64.4)	209 (71.8)	0.214
Smoking (%)	111 (30.5)	18 (24.7)	93 (32.0)	0.226
Coronary artery disease (%)	72 (19.8)	19 (26.0)	53 (18.2)	0.134
Hyperlipidemia (%)	295 (81.0)	61 (83.6)	234 (80.4)	0.539
Hypertension (%)	224 (61.5)	49 (67.1)	175 (60.1)	0.273
Retinopathy (%)	34 (9.3)	15 (20.5)	19 (6.5)	<0.001
Neuropathy (%)	33 (9.1)	12 (16.4)	21 (7.2)	0.014
Albuminuria (%)	121 (33.2)	36 (49.3)	85 (29.2)	0.001
CKD stage 3 (%)	67 (18.4)	21 (28.8)	46 (15.8)	0.011
CKD stage 3 with albuminuria (%)	41 (11.3)	16 (21.9)	25 (8.6)	0.001
Duration of diabetes (year)	11.7±7.5	14.7±9.1	10.9±6.9	0.001
Body mass index	26.3±4.4	26.4±5.6	26.3±4.1	0.842
Waist-hip ratio	0.94±0.07	0.94±0.07	0.94±0.06	0.858
Systolic blood pressure (mmHg)	132.8±15.4	134.9±16.5	132.3±15.1	0.204
Diastolic blood pressure (mmHg)	77.3±10.7	78.4±12.0	77.0±10.3	0.299
Mean blood pressure (mmHg)	114.0±13.9	116.0±13.8	113.9±12.2	0.183
Urinary albumin-creatinine ratio (mg/g Cr)	104.8±301.9	258.1±521.9	64.7±191.7	0.003
HbA1C (%)	7.1±0.8	7.2±0.8	7.0±0.8	0.118
Total cholesterol (mg/dL)	166.8±28.1	167.1±26.0	166.7±28.7	0.914
Creatinine (mg/dL)	0.97±0.27	1.01±0.30	0.95±0.26	0.116
eGFR (mL/min/1.73m2)	78.2±19.4	72.6±20.4	79.6±19.0	0.006
sTNFR2 (ng/mL)	2.00±1.17	2.35±1.15	1.92±1.15	0.004
Metformin (%)	283 (77.7)	56 (76.7)	227 (78.0)	0.812
Sulfonylurea (%)	144 (39.6)	32 (43.8)	112 (38.5)	0.403
Dipeptidyl peptidase-4 inhibitor (%)	68 (18.7)	17 (23.3)	51 (17.5)	0.259
Sodium/glucose co-transporter 2 inhibitor (%)	13 (3.6)	1 (1.4)	12 (4.1)	0.257
Insulin (%)	64 (17.6)	17 (23.3)	47 (16.2)	0.152
Renin-angiotensin system blockade (%)	174 (47.8)	45 (61.6)	129 (44.3)	0.008
Diuretics (%)	43 (11.8)	15 (20.5)	28 (9.6)	0.010

Data are expressed as mean±SD for continuous variables and numbers and percentages for non-continuous variables. Differences between groups stratified by the occurrence of renal outcomes were analyzed by Student’s t-test for continuous variables and Pearson’s chi-squared test for non-continuous variables.

CKD, chronic kidney disease; Cr, creatinine; HbA1C, hemoglobin A1C; eGFR, estimated glomerular filtration rate; sTNFR2, soluble tumor necrosis factor receptor type 2.

Association of sTNFR2 with the renal outcomes

Over 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 (Table 1). Correlations between sTNFR2 concentrations and BMIs or waist-to-hip ratios were weak (Pearson’s correlation coefficient -0.062 for sTNFR2 and BMI, p = 0.241; -0.021 for sTNFR2 and waist-hip-ratio, p = 0.686). The sTNFR2 concentration was associated with renal outcomes in the univariate Cox proportional analysis (hazard ratio [HR] 1.29, 95% confidence interval [CI] 1.09–1.52, p = 0.003). Other clinical covariates, such as the duration of diabetes, presence of retinopathy or neuropathy, eGFR and UACR at baseline, and the use of RAS inhibitors or diuretics were also associated with renal outcomes. The association of sTNFR2 concentration with renal events was attenuated after adjustment in sequential models (Table 2).

Table 2

Results of the univariate and multivariate Cox proportional hazard models for the association of sTNFR2 with renal composite events.

	HR	95% CI	P value
sTNFR2	1.29	1.09–1.52	0.003
sTNFR2 ≥1.608 ng/mL	2.47	1.45–4.21	0.001
Duration of diabetes	1.05	1.02–1.08	<0.001
Retinopathy	2.78	1.58–4.92	<0.001
Neuropathy	1.86	1.00–3.46	0.049
eGFR	0.98	0.97–1.00	0.009
UACR (per 100 mg/g Cr increase)	1.10	1.06–1.14	<0.001
RAS inhibitors	1.73	1.08–2.77	0.023
Diuretics	1.78	1.01–3.15	0.046
Model 1 for sTNFR2	1.27	1.07–1.52	0.007
Model 2 for sTNFR2	1.24	1.03–1.50	0.023
Model 3 for sTNFR2	1.11	0.88–1.41	0.394
Model 1 for sTNFR2 ≥1.608 ng/mL	2.41	1.38–4.21	0.002
Model 2 for sTNFR2 ≥1.608 ng/mL	2.31	1.32–4.04	0.003
Model 3 for sTNFR2 ≥1.608 ng/mL	2.27	1.23–4.20	0.009

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.

HR, hazard ratio; CI, confidence interval; sTNFR2, soluble tumor necrosis factor receptor type 2; eGFR, estimated glomerular filtration rate; UACR, urinary albumin-creatinine ratio; Cr, creatinine; RAS, renin-angiotensin system; HbA1C, hemoglobin A1C.

The 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 (95% CI 0.57–0.70, p < 0.001; Fig 1). The highest tertile of sTNFR2 concentration had an increased risk of renal composite events (p = 0.012 from the log-rank test; Fig 2a).

Fig 1

Receiver operating characteristic curves of soluble tumor necrosis factor receptor 2 (sTNFR2) in patients with type 2 diabetes.

Fig 2

Kaplan-Meier curves of cumulative event-free survival of renal events (a composite of a >30% reduction of the estimated glomerular filtration rate and/or the progression of albuminuria stages) in patients with type 2 diabetes according to: (a) the tertiles of soluble tumor necrosis factor receptor 2 (sTNFR2) levels and (b) the threshold value of sTNFR2 (≥1.608 ng/mL vs. <1.608 ng/mL).

Differences between curves were analyzed with log-rank statistics.

Association of sTNFR2 levels ≥1.608 ng/mL with the renal outcomes

The clinical characteristics of the study population stratified based on sTNFR2 concentrations (≥1.608 ng/mL or <1.608 ng/mL) are shown in S1 Table. The patients with sTNFR2 concentrations ≥1.608 ng/mL were older and had longer durations of diabetes, higher percentage of retinopathy, and higher UACRs and lower eGFRs at baseline. The coverage of the RAS blockade was more extensive in patients with sTNFR2 concentrations ≥1.608 ng/mL than in patients with sTNFR2 concentrations <1.608 ng/mL (55.3% vs. 38.9%, p = 0.002). The patients with sTNFR2 concentrations ≥1.608 ng/mL had more renal composite events at the end of the study than those with sTNFR2 concentrations <1.608 ng/mL did (27.9% vs. 10.8%; p < 0.001 from the log-rank test; Fig 2b). Regarding the separate components of the renal composite events, the frequency of each was also higher in the study group with sTNFR2 concentrations ≥1.608 ng/mL (13.7% vs 6.6% for eGFR decline >30%, p = 0.027; 18.3% vs. 6.6% for worsening albuminuria, p = 0.001; S1 Table).

In the univariate Cox proportional analysis, sTNFR2 concentrations ≥1.608 ng/mL were associated with higher renal event rates (HR 2.47, 95% CI 1.45–4.21, p = 0.001). A sTNFR2 concentration ≥1.608 ng/mL predicted renal events in all of the multivariate models (HR 2.41, 95% CI 1.38–4.21, p = 0.002 for model 1; HR 2.31, 95% CI 1.32–4.04, p = 0.003 for model 2; HR 2.27, 95% CI 1.23–4.20, p = 0.009 for model 3; Fig 3).

Fig 3

Cumulative hazard curves for renal events (a composite of a >30% reduction of the estimated glomerular filtration rate and/or the progression of albuminuria stages) 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.

We 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 (Crude HR 3.35, 95% CI 1.15–9.77, p = 0.027; HR 3.53, 95% CI 1.19–10.49, p = 0.023 for model 1; HR 3.00, 95% CI 0.98–9.15, p = 0.053 for model 2; HR 4.12, 95% CI 1.00–17.07, p = 0.051 for model 3).

Table 3

Results of the association between sTNFR2 concentrations ≥1.608 ng/mL and renal composite events stratified by age, sex, systolic blood pressure, eGFR, UACR, and the use of RAS inhibitors.

Subgroup	Crude HR	95% CI	P value for interaction
Age ≥60 years old (n = 222)	2.58	1.16–5.76	0.87
Age <60 years old (n = 142)	2.63	1.19–5.81
Male sex (n = 256)	2.12	1.13–3.97	0.19
Female sex (n = 108)	3.35	1.15–9.77
SBP ≥140 mmHg (n = 110)	3.49	1.19–10.26	0.62
SBP <140 mmHg (n = 254)	2.17	1.17–4.03
eGFR ≥60 ml/min/1.73m2 (n = 297)	2.64	1.45–4.82	0.39
eGFR <60 ml/min/1.73m2 (n = 67)	0.84	0.25–2.85
UACR ≥30 mg/g Cr (n = 121)	1.91	0.79–4.60	0.27
UACR <30 mg/g Cr (n = 243)	2.38	1.19–4.75
RAS inhibitors (n = 174)	2.76	1.28–5.93	0.19
No RAS inhibitors (n = 190)	1.90	0.87–4.12

HR, hazard ratio; CI, confidence interval; SBP, systolic blood pressure; eGFR, estimated glomerular filtration rate; UACR, urinary albumin-creatinine ratio; Cr, creatinine; RAS, renin-angiotensin system.

Discussion

In 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.

In addition to the traditional risk factors, such as poor glycemic [14] or blood pressure [15] control, chronic low-grade inflammation plays an important role in the development of DKD [8]. Activation of the tumor necrosis factor-α pathways is associated with renal function decline in patients with chronic kidney disease [16]. Tumor necrosis factor-α is involved in the signal transduction pathways by interacting with two types of membrane receptors: TNFR 1 and TNFR2 [17]. Tumor necrosis factor receptor 1 is mainly present in glomerular and tubular endothelial cells, whereas TNFR2 is usually absent in normal kidneys and transcriptionally expressed in the renal cells in various kidney diseases [18]. Tumor necrosis factor receptor 1 and TNFR2 induce shared and distinctive signalling pathways that lead to apoptosis, proliferation, and inflammation [8]. Both membrane-bound receptors TNFR1 and TNFR2 can be cleaved by the metalloproteinase and released as circulating polypeptides, soluble TNFR1 (sTNFR1) and sTNFR2 [19]. Higher 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.

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.

Soluble tumor necrosis factor receptor 1 has been considered a valuable predictor of renal function decline among patients with type 2 diabetes [10–13, 21]. A recent meta-analysis demonstrated that both circulating sTNFR1 and sTNFR2 levels were independently associated with DKD progression in more than 5000 patients with diabetes [22]. We 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 (HR 2.43, 95% CI 1.18–5.02, p = 0.01). 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.

Patients with type 2 diabetes and renal impairments do not always have preceding albuminuria [23]. In the United Kingdom Prospective Diabetes Study (UKPDS) cohort, 51% of the patients with renal impairments had normoalbuminuria for a median of 15 years of follow-up [24]. The presence of nonproteinuric renal dysfunction in patients with type 2 diabetes increases their risk of ESRD and cardiovascular disease [25]. Increased vascular resistance in renal interlobar arteries has been suggested to cause damage to nephron structures, contributing to nonproteinuric diabetic nephropathy [26]. Tumor necrosis factor-α is an essential mediator of inflammation that can induce renal vasoconstriction, which is a potential mechanism of nonproteinuric diabetic nephropathy [27]. In our study, the association of serum sTNFR2 levels with renal composite events was consistent across multiple prespecified subgroups, including participants with and without albuminuria. This finding identified sTNFR2 as a potential predictor of renal function decline in patients with type 2 diabetes and normoalbuminuria.

There may be a racial disparity in serum sTNFR2 levels among patients with type 2 diabetes. The median serum sTNFR2 level in Pima Indians (4835 pg/mL) [12] was much higher than that in Caucasian patients in the Joslin Study of the Genetics of Type 2 Diabetes and Kidney Complications (2527 pg/mL) [11], although increased serum sTNFR2 levels in Pima Indians may be partly attributed to obesity. In a previous study comprising of Japanese patients [28], the median serum sTNFR2 level in participants with an eGFR ≥60 mL/min/1.73m2 was 2972 pg/mL, which was comparable with that in Caucasians. The serum sTNFR2 levels in our study did not seem markedly different from those of previous studies, and the results from our cohort still imply that sTNFR2 could be a potential predictor of renal outcomes in patients with type 2 diabetes. Further studies are needed to clarify the normal ranges among different races.

The 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.

Conclusions

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.

Baseline characteristics and renal composite events of patients grouped according to soluble tumor necrosis factor receptor type 2 (sTNFR2) concentrations.

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31 Jan 2022

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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.

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?

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Reviewers' comments:

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #2: Yes

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5. 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. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Wu 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 ( increased albuminuria and reduced egfr). 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?

**********

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Reviewer #1: No

Reviewer #2: No

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3 Mar 2022

Academic Editor

1. 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 (for instance, written or verbal, and if verbal, how it was documented and witnessed). 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”

Original

This 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.

Revised

This 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 letter

Original

This 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.

Revised

This 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”

Original

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. 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.

Revised

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.

Cover letter

Original

This 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.

Revised

This 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 (doi: 10.4158/EP-2020-0114, PMID: 32576039). 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 (95% CI 0.57-0.71, p < 0.001) for sTNFR1 and 0.63 (95% CI 0.57-0.70, p < 0.001) 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”

Original

In 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.

Revised

In 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”

Original

We 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 (HR 2.43, 95% CI 1.18-5.02, p = 0.01). 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.

Revised

We 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 (HR 2.43, 95% CI 1.18-5.02, p = 0.01). 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 1

Wu 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 (increased albuminuria and reduced egfr). 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 (doi: 10.4158/EP-2020-0114, PMID: 32576039). 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”

Original

We previously reported that serum sTNFR1 levels were associated with renal outcomes in patients with type 2 diabetes [9].

Revised

We 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 (95% CI 0.57-0.71, p < 0.001) for sTNFR1 and 0.63 (95% CI 0.57-0.70, p < 0.001) 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”

Original

We 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 (HR 2.43, 95% CI 1.18-5.02, p = 0.01). 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.

Revised

We 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 (HR 2.43, 95% CI 1.18-5.02, p = 0.01). 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”

Original

In the univariate Cox proportional analysis, sTNFR2 concentrations ≥1.608 ng/mL were associated with higher renal event rates (HR 2.47, 95% CI 1.45-4.21, p = 0.001). A sTNFR2 concentration ≥1.608 ng/mL predicted renal events in all of the multivariate models (HR 2.41, 95% CI 1.38-4.21, p = 0.002 for model 1; HR 2.31, 95% CI 1.32-4.04, p = 0.003 for model 2; HR 2.27, 95% CI 1.23-4.20, p = 0.009 for model 3).

Revised

In the univariate Cox proportional analysis, sTNFR2 concentrations ≥1.608 ng/mL were associated with higher renal event rates (HR 2.47, 95% CI 1.45-4.21, p = 0.001). A sTNFR2 concentration ≥1.608 ng/mL predicted renal events in all of the multivariate models (HR 2.41, 95% CI 1.38-4.21, p = 0.002 for model 1; HR 2.31, 95% CI 1.32-4.04, p = 0.003 for model 2; HR 2.27, 95% CI 1.23-4.20, p = 0.009 for model 3; Fig 3).

Fig 3.

Cumulative hazard curves for renal events (a composite of a >30% reduction of the estimated glomerular filtration rate and/or the progression of albuminuria stages) 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 (Crude HR 3.35, 95% CI 1.15-9.77, p = 0.027; HR 3.53, 95% CI 1.19-10.49, p = 0.023 for model 1; HR 3.00, 95% CI 0.98-9.15, p = 0.053 for model 2; HR 4.12, 95% CI 1.00-17.07, p = 0.051 for 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.

We revise the manuscript as below:

Line 270, page 20, section of “Discussion”

Original

Higher 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.

Revised

Higher 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”

Original

We 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).

Revised

We 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 (Crude HR 3.35, 95% CI 1.15-9.77, p = 0.027; HR 3.53, 95% CI 1.19-10.49, p = 0.023 for model 1; HR 3.00, 95% CI 0.98-9.15, p = 0.053 for model 2; HR 4.12, 95% CI 1.00-17.07, p = 0.051 for model 3).

Line 320, page 23, section of “Discussion”

Original

The 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.

Revised

The 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”

Original

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.

Revised

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.

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”

Original

Higher 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.

Revised

Higher 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”

Original

In 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.

Revised

In 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.

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29 Mar 2022

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

PONE-D-21-32495R1

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Thank you for responding to the queries. Please amend title: Soluble tumour necrosis factor receptor 2 "is"

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Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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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.

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Reviewer #2: No

1 Apr 2022

PONE-D-21-32495R1

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

Dear Dr. Lin:

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