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Tissue expression of tubular injury markers is associated with renal function decline in diabetic nephropathy
Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
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Tissue expression of tubular injury markers is associated with renal function decline in diabetic nephropathy Subin Hwang a, Jeeeun Park a, Jinhae Kim a, Hye Ryoun Jang a, Ghee Young Kwon b, Wooseong Huh a, Yoon-Goo Kim a, Dae Joong Kim a, Ha Young Oh a, Jung Eun Lee a,⁎ a b
Nephrology Division, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 10 May 2017 Received in revised form 28 July 2017 Accepted 20 August 2017 Available online xxxx Keywords: Diabetic nephropathy Diabetic kidney disease Neutrophil gelatinase-associated lipocalin Kidney injury molecule-1 Immunohistochemistry
a b s t r a c t Aims: The pathogenesis of diabetic kidney disease (DKD) is complex and multifactorial; increasing evidence suggests that tubular injury and inflammatory process are involved in disease progression. We investigated the potential association of renal expression of tubular injury markers, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and inflammatory markers, tumor necrosis factor receptor (TNFR) 1 and 2 with renal progression in pathologically proven diabetic nephropathy (DN). Methods: We identified 122 patients with confirmed DN. After excluding patients with other coexisting renal disease or estimated glomerular filtration rate (eGFR) b 30 mL/min/1.73 m2, 35 patients were included. Annual decline of (GFR decline slope) was calculated using linear regression analysis. Tissue tubular and glomerular expressions of NGAL, KIM-1, TNFR1, and TNFR2 were assessed using immunohistochemistry. Results: Median baseline urinary protein to creatinine ratio (uPCR) was 6.76 (2.18–7.61) mg/mg Cr, median baseline eGFR was 50 (43–66) mL/min per 1.73 m2, and median GFR decline slope was 15.6 (4.4–35.1) mL/ min per 1.73 m2 per year. Positive correlations were observed between tubular expressions of NGAL and KIM-1, and GFR decline slopes (r = 0.601, p b 0.001; r = 0.516, p = 0.001, respectively), and between tubular expressions of KIM-1 and uPCR (r = 0.596, p b 0.001), and between NGAL and interstitial fibrosis and tubular atrophy (IFTA) score (r = 0.391, p = 0.024). No correlations were found between glomerular or tubular expressions of TNFRs, and clinical parameters including GFR decline slopes. On multivariate analysis, the association between tubular expressions of KIM-1 and GFR decline slopes was dependent on uPCR. Tubular expressions of NGAL were independently associated with GFR decline slopes, with an adjusted coefficient factor of 0.290 (95% confidence interval, 0.009–0.202, p = 0.038). Conclusions: These findings suggest that tubular injury plays a key role in the pathogenesis of DKD in high-risk patients. Further studies are warranted to determine whether tubular injury could be a therapeutic target in DKD. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Despite major implementation of renoprotective treatment in recent decades, diabetic kidney disease (DKD) continues to rank as the leading cause of end-stage renal disease (ESRD). Paradoxically, improvements in cardiovascular outcomes in patients with diabetes have allowed ample time for the development of kidney dysfunction and ESRD. 1 Therefore, prevention and delay of DKD progression is becoming increasingly important to reduce public health burdens. Conflict of Interest Statement: All authors declare that they have no conflict of interest. ⁎ Corresponding author at: Division of Nephrology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, 06351, Seoul, Republic of Korea. E-mail address: [email protected] (J.E. Lee).
Susceptibilities to DKD and outcomes are highly variable. The amount of proteinuria, elevated blood pressure (BP), decreased kidney function, hyperglycemia, and episodes of acute kidney injury (AKI) have been identified as risk factors for the progression of DKD. 2–10 Classically, glomeruli have been considered the primary injury site for diabetic nephropathy (DN), and albuminuria, which reflects glomerular damage, has been a key prognostic marker. Recently, several biomarkers related to inflammation or tubular injury have been identified as potent predictors of renal outcome. Circulating tumor necrosis factor receptor (TNFR) 1 and 2 have been reported to be strongly associated with subsequent progression to ESRD in patients with diabetes.11,12 Furthermore, urinary and plasma levels of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) may function as early markers of DN. 13–16
https://doi.org/10.1016/j.jdiacomp.2017.08.009 1056-8727/© 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Hwang S, et al. Tissue expression of tubular injury markers is associated with renal function decline in diabetic nephropathy. Journal of Diabetes and Its Complications (2017), https://doi.org/10.1016/j.jdiacomp.2017.08.009
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S. Hwang et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
Accumulating evidence indicates that the inflammatory process and tubular damage are early events in the course of DN and are not merely secondary to glomerular damage.17–19 In the present study, we investigated whether renal expression of NGAL, KIM-1, TNFR1, and TNFR2 predict subsequent decline of kidney function in subjects with pathologically proven DN. 2. Materials and methods 2.1. Population We identified 122 patients with diabetes mellitus (DM) who underwent renal biopsy and were confirmed to have DN between January 2000 and December 2014 at a 2000-bed tertiary referral center in Seoul, Korea. We excluded subjects with other coexisting renal disease based on pathologic findings (other types of glomerulonephritis, acute tubular necrosis, or acute interstitial nephritis) (n = 24), those with estimated GFR (eGFR) b30 mL/min/1.73 m 2 at the time of biopsy (n = 54), those who received treatment with immunosuppressants (n = 2), and those who underwent follow-up for b 6 months or b3 visits after renal biopsy (n = 7). Finally, 35 subjects were included in the analyses. All included subjects had provided written informed consent at the time of biopsy for the collection and use of their remnant biopsy specimens, and serum and urinary samples for research regarding biomarkers. This study was approved by the institutional review board of Samsung Medical Center. As this study was retrospective and the subjects were de-identified, the institutional review board waived the need for additional written consent from the subjects. 2.2. Clinical and laboratory data Data regarding age, sex, duration and type of diabetes, presence of diabetic retinopathy, anti-hypertensive treatment including angiotensin-converting enzyme inhibitor (ACE-I) or angiotensin receptor blocker (ARB), body mass index (BMI), smoking status, and systolic and diastolic (BP) at the time of renal biopsy were obtained from electronic medical records of the patients. Hemoglobin A1c (HbA1c) levels at the time of renal biopsy were also obtained. The average of the urinary protein to creatinine ratio (uPCR) in spot urine samples obtained during the 6 months after renal biopsy was used as the baseline time-averaged uPCR. The primary outcome was the GFR decline slope (in mL/min/1.73 m 2 per year). To estimate the GFR decline slopes for each patient, we collected all serum creatinine measurements from 3 months before renal biopsy to the day of the last follow-up. We then calculated eGFR using the Chronic Kidney Disease Epidemiology Collaboration formula. 20 Next, a model for time versus eGFR was created using a linear regression analysis, and the absolute values of the slope of the regression line were regarded as GFR decline slopes over time, with a positive value representing a decreasing trajectory of GFR. 2.3. Renal biopsy and pathologic classification Kidney histology was retrospectively reviewed. The tissues had been obtained by needle biopsy, and the specimens had been processed for light microscopy and immunofluorescence, as well as for electron microscopy. Classification of DN and histological scoring were performed according to the criteria of Mooyaar et al. 21 Renal pathological findings were also included as categorical covariates: glomerular class (I, IIa, IIb, III, IV); interstitial fibrosis and tubular atrophy (IFTA) score (0, 1, 2, 3); interstitial inflammation score (0, 1, 2); arteriolar hyalinosis score (0, 1, 2); and arteriosclerosis score (0, 1, 2).
2.4. Immunohistochemical (IHC) study Serial sections of formalin-fixed paraffin-embedded kidney tissues were prepared and placed on a microscope slide. After deparaffinization and rehydration, tissue sections were washed, followed by blocking of endogenous peroxidase through incubation with Dako REAL Peroxidase Blocking solution (DAKO, Seoul, South Korea) for 30 min at room temperature. In addition, tissues prepared for NGAL were incubated with avidin and biotin for 15 min. Then, all tissues were treated with Dako Protein Block Serum-Free agent (DAKO) overnight at 4 °C to prevent background staining. Subsequently, tissue sections were incubated in primary antibodies KIM1 (Human TIM-1/KIM-1/HAVCR Ab; R&D Systems, Boston, MA, USA; 1:300), NGAL (Human Lipocalin-2/NGAL Antibody; R&D Systems; 1:100), and TNFR1 and TNFR2 (Anti-TNF Receptor I Ab and II Ab; Abcam, Cambridge, UK; 1:500 and 1:50, respectively) for 1 h at room temperature. Thereafter, the tissue sections were incubated with a secondary antibody (EnVision for KIM-1, TNFR1, and TNFR2; rabbit polyclonal antibody to rat I immunoglobulin G-biotin for NGAL) for 30 min. Then, streptavidin horseradish peroxidase (HRP) for tissues that reacted with NGAL antibody was added, along with 3,3′ diaminobenzidine (DAB) chromogenic for visualization in a color reaction, followed by hematoxylin dye. Brown staining showed positive results. Staining levels were scored as follows. The IHC staining was evaluated by the percentage and intensity of positive tubular or glomerular cells. The percentage of positive cells was recorded as 0 (0–10%), 1 (11–25%), 2 (26–50%), 3 (51%–75%), or 4 (N 75%). The staining intensity of positive cells was recorded as 0 (negative), 1 (weakly positive), 2 (moderately positive), or 3 (strongly positive). The total score (0−12) was calculated by multiplying the two parameters. Positive immunoreactivity for NGAL and KIM-1 was predominantly detected in renal tubules, while immunoreactivity for TNFR1 and TNFR2 was detected in both tubules and glomeruli. Therefore, the glomerular and tubular expressions of TNFRs were separately assessed. 2.5. Serum and urine NGAL and KIM-1 measurements Serum NGAL (sNGAL), serum KIM-1 (sKIM-1), urinary creatinine, urinary NGAL (uNGAL), and urinary KIM-1 (uKIM-1) concentrations were measured using enzyme-linked immunosorbent assay (ELISA) kits (NGAL: Lipocalin-2/NGAL, R&D Systems; KIM-1: TIM-1/KIM-1/ HAVCR, R&D Systems), according to the manufacturer's instructions. Measurements were performed in duplicate and triplicate for serum and urine, respectively. 2.6. Statistical analyses Data were expressed as percentages for categorical variables and as medians (interquartile ranges, IQRs) for continuous variables. As some variables (GFR decline slope, HbA1c, and uPCR) showed skewed distributions, log transformation was carried out before performing correlation and regression analysis. The Mann–Whitney rank sum test was used for comparisons of continuous variables, and Fisher's exact test was used for categorical variables. Correlations among continuous variables were assessed using the Spearman rank correlation coefficient. To investigate the variables that could predict rapid progression of DN, multivariate linear regression analysis was conducted to explore the association of GFR decline slopes with baseline clinical and histopathological variables, and tubular expression of NGAL and KIM-1 following univariate linear regression with p b 0.2, and the significant explanatory parameters were chosen in an enter stepwise manner. Results were expressed as the regression coefficient (β).
Please cite this article as: Hwang S, et al. Tissue expression of tubular injury markers is associated with renal function decline in diabetic nephropathy. Journal of Diabetes and Its Complications (2017), https://doi.org/10.1016/j.jdiacomp.2017.08.009
S. Hwang et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
Statistical significance was defined as p b 0.05. All statistical analyses were performed using SPSS for Windows, version 23.0 (IBM Corp., Chicago, IL, USA).
3. Results Of the 35 subjects, 28 were men (80%). At the time of biopsy, the median age of the subjects was 50 (43–59) years (Table 1). The duration of DM was 105–14 years, and 83% of the subjects had diabetic retinopathy. The median follow-up duration after biopsy was 24.2 (13.5–51.2) months. The median time-averaged uPCR was 6.76 (2.18–7.61) mg/mg Cr and the median baseline eGFR was 50 (43–66) mL/min/1.73 m 2. Based on the glomerular classification, 1 (3%) subject was in class I, 2 subjects (6%) in class IIa, 4 (11%) in class IIb, 24 (69%) in class III, and 4 (11%) in class IV. IFTA scores of 0, 1, 2 and 3 were observed in 2 (6%), 20 (57%), 12 (34%), and 1 (3%) subjects, respectively. Interstitial inflammation of scores 0, 1, and 2 was observed in 2 (6%), 30 (86%), and 3 (8%) subjects, respectively. Arteriolar hyalinosis was absent in 5 subjects (14%). More than one area of arteriolar hyalinosis (scored as 2) was found in 28 subjects (80%). In 2 subjects, large vessels were absent in renal biopsies. Among the remaining 33 subjects, 6 (18%) had no intimal thickening (scored as 0) and 14 (42%) had severe arteriosclerosis (scored as 2). The median GFR decline slope was 15.6 (4.4–35.1) mL/min/1.73 m 2 per year. Evaluation of the correlation of pathologic classification with clinical parameters (Table 2) showed that IFTA scores were positively correlated with GFR decline slopes (r = 0.415, p = 0.024) and
Table 1 Baseline clinical and pathologic characteristics. Variables
n = 35
Age (years) Female, n, (%) Follow-up duration (months) Diabetes duration (years) Diabetes type, n (%) Type 1/type 2 Diabetic retinopathy, n (%) Body mass index (kg/m2) Smoking status, n (%) Never/ex-/current smoker Systolic BP (mm Hg) Diastolic BP (mm Hg) ACE-I or ARB treatment, n (%) Hemoglobin A1c (%) uPCR (mg/mg Cr) CKD stage, n (%) 1/2/3A/3B eGFR (mL/min/1.73 m2) GFR decline slope (mL/min/1.73 m2 per year) Glomerular class, n (%) I/IIa/IIb/III/IV IFTA, n (%) 0/1/2/3 Interstitial inflammation score, n (%) 0/1/2 Arteriolar hyalinosis score, n, (%) 0/1/2 Arteriosclerosis scorea, n (%) 0/1/2
50 (43–59) 7 (20%) 24.2 (13.5–51.2) 10 (5–14) 2 (6%)/33 (94%) 29 (83%) 23.6 (22.4–25.5) 21 (60%)/5 (14%)/9 (26%) 139 (132–150) 80 (73–89) 28 (80%) 7.3 (6.0–8.2) 6.76 (2.18–7.61) 4 (11%)/7 (20%)/12 (34%)/12 (34%) 50 (43–66) 15.6 (4.4–35.1)
1 (3%)/2 (6%)/4 (11%)/24 (69%)/4 (11%) 2 (6%)/20 (57%)/12 (34%)/1 (3%) 2 (6%)/30 (86%)/3 (8%) 5 (14%)/2 (6%)/28 (80%) 6 (18%)/13 (39%)/14 (42%)
Continuous variables were expressed as median (interquartile range). Abbreviations: BP, blood pressure; ACE-I, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; uPCR, urinary protein to creatinine ratio; CKD, chronic kidney disease; eGFR, estimated glomerular filtration rate; IFTA, interstitial fibrosis and tubular atrophy. a Arteriosclerosis score was missing for two subjects because their renal biopsy specimen did not include any arteries.
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arteriolar hyalinosis scores were correlated with uPCR (r = 0.399, p = 0.018). Additionally, negative and positive correlations were observed, respectively, between interstitial inflammation scores and BMI (r = −0.336, p = 0.048), and between arteriosclerosis scores and diabetes duration (r = 0.359, p = 0.040). Tissue expression of NGAL, KIM-1, TNFR1, and TNFR2 was examined in each patient (Fig. 1). The correlations between tissue expressions of NGAL, KIM-1, TNFR1, and TNFR2, and clinical and pathologic parameters are presented in Table 3. There were positive correlations between tubular expressions of NGAL and KIM-1, and GFR decline slopes (r = 0.601, p b 0.001; r = 0.516, p = 0.001, respectively). Moreover, the expressions of KIM-1 and uPCR (r = 0.596, p b 0.001) and the expressions of NGAL and IFTA scores (r = 0.391, p = 0.024) showed positive correlations. However, no correlations were observed between glomerular or tubular expressions of TNFRs and clinical parameters including GFR decline slopes. When examining the renal expressions of NGAL and KIM-1 according to treatment status with ACE-I or ARB, the median score of tubular expressions of NGAL was 5 4–6 in the group treated with ACE-I or ARB and 3 3–5 in the group not treated with ACE-I or ARB. The median score of tubular expressions of KIM-1 was 6 (3–7.8) in the group treated with ACE-I or ARB, and it was 5 4–8 in the others. There were no significant differences between the groups (p = 0.444 and p = 0.806, respectively). To identify parameters to predict the GFR decline slope, linear regression analyses were conducted. Among the clinical parameters, diabetes duration, BMI, and uPCR were associated with GFR decline slopes (β = 0.026, p = 0.184 for diabetes duration; β = 0.926, p b 0.001 for uPCR; and β = −0.079, p = 0.012 for BMI, respectively) in univariate analyses. Multivariate analysis revealed that the association between tubular expressions of KIM-1 and GFR decline slopes was dependent on uPCR. In contrast, tubular expressions of NGAL demonstrated an independent association with GFR decline slopes, with an adjusted coefficient factor of 0.290 (95% confidence interval [CI], 0.009–0.202, p = 0.038) (Table 4). For analyses of NGAL and KIM-1 concentrations, serum samples were available in 14 subjects and urine samples were available in 18 subjects. Tubular expressions of KIM-1 were not correlated with sKIM-1 (r = 0.366, p = 0.135), but those were highly correlated with uKIM-1 (r = 0.878, p b 0.001). Tubular expressions of NGAL showed a positive correlation with uNGAL (r = 0.529, p = 0.052) (Table 5). 4. Discussion In the present study, we evaluated the relationship between the tissue expressions of NGAL, KIM-1, TNFR1 and TNFR2, clinical and histopathologic parameters, and renal progression in subjects with pathologically proven DN. Proteinuria is a major clinical determinant of subsequent GFR decline slopes, and tubulointerstitial change is the most important histologic finding associated with the progression of DN. Renal expressions of TNFR1 and TNFR2 were not associated with proteinuria and baseline GFR or with GFR decline slope in the current study. Meanwhile, increased tubular expressions of NGAL and KIM-1 were associated with subsequent GFR decline. Moreover, the association of tubular expressions of NGAL and GFR decline slopes was independent of proteinuria and tubulointerstitial change. Considering that this study included patients with rapidly progressive DN, these findings suggest that tubular injury plays a key role in the pathogenesis of DN in patients at high risk. KIM-1 and NGAL are well-known tubular injury markers. It is now increasingly recognized that tubular injury has an important role in the pathogenesis and progression of DN, and the severity of tubulointerstitial lesions has a significant impact on renal outcome in DN. 22,23 The mechanisms that underlie tubular injury are
Please cite this article as: Hwang S, et al. Tissue expression of tubular injury markers is associated with renal function decline in diabetic nephropathy. Journal of Diabetes and Its Complications (2017), https://doi.org/10.1016/j.jdiacomp.2017.08.009
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S. Hwang et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
Table 2 Correlation of pathologic classification with clinical parameters. Variables
Age Diabetes duration BMI Smoking status Hemoglobin A1c eGFR uPCR GFR decline slope
r Glomerular class
IFTA score
Interstitial inflammation score
Arteriolar hyalinosis score
Arteriosclerosis score
0.012 0.009 −0.147 −0.352⁎ 0.180 −0.008 −0.005 0.025
−0.291 −0.017 −0.029 −0.033 0.103 −0.094 0.244 0.415⁎
−0.007 −0.020 −0.336⁎
−0.203 −0.016 0.023 0.224 0.321 0.166 0.399⁎ 0.293
0.313 0.359⁎ −0.002 0.192 0.146 −0.073 0.056 0.078
−0.289 0.073 −0.033 0.203 0.034
Abbreviations: IFTA, interstitial fibrosis and tubular atrophy; BMI, body mass index; BP, blood pressure; eGFR, estimated glomerular filtration rate; uPCR, urinary protein to creatinine ratio. ⁎ p-Value b 0.05.
complex. 18 Tubular epithelial cells are persistently exposed to hyperglycemic conditions by tubular reabsorption of glucose in patients with diabetes.19 In addition, the local renin–angiotensin system and chronic hypoxia contribute to progressive tubular injury. Furthermore, albuminuria may be directly harmful to renal tubular cells, leading to tubular inflammation and fibrosis.24 The proximal tubule, in particular, is more vulnerable to these metabolic and hemodynamic factors. Brito et al. 25 have shown that the proximal tubular basement membrane is already thickened in normoalbuminuric patients with diabetes. In addition, a few studies have demonstrated that excessive iron deposit can lead to increased oxidative stress injury in proximal tubule, leading to cell damage. 26 The present study is, to the best of our knowledge, the first to demonstrate, using human histology, that increased tissue expressions of tubular injury markers are associated with progressive loss of kidney function. Further studies
are needed to evaluate whether modulation of tubular injury could be a target of new therapeutic intervention in DN. Serum and urinary NGAL levels have been studied as potential biomarkers of DKD, demonstrating correlations with GFR and/or albuminuria.27–30 However, the prognostic power of these markers to predict the progression of DKD has not exceeded the power of clinical parameters. 31 One limitation of serum or urine NGAL levels as biomarkers is that the NGAL protein is not specific to kidney injury. In other words, the protein can be released into the circulation from any organs with inflammation. Furthermore, serum NGAL can be filtered in the glomerulus, so serum levels are highly dependent on the current glomerular filtration rate, even in circumstances other than those of kidney injury. Thus, serum and urinary levels of NGAL may not directly reflect intrarenal NGAL expression in patients with CKD. The present study evaluated the tubular expression of NGAL in
Fig. 1. Tissue expression of NGAL, KIM-1, and TNFR1 and TNFR2 in kidneys with diabetic nephropathy. Representative images of immunohistochemistry (IHC)-stained section of the kidney with diabetic nephropathy (original magnification, ×200). IHC sections for NGAL (A–D), KIM-1 (E–H), TNFR1 (I–K), and TNFR2 (L–O). Percentage of positive cells was recorded as 0 (0–10%), 1 (11–25%), 2 (26–50%), 3 (51–75%), or 4 (N75%), and staining intensity was recorded as 0 (negative), 1 (weakly positive), 2 (moderately positive), or 3 (strongly positive). Total score (0–12) was calculated by multiplying the two parameters. There was no specimen compatible with “negative” for TNFR1. Abbreviations: NGAL, neutrophil gelatinase-associated lipocalin; KIM-1, kidney injury molecule-1; TNFR, tumor necrosis factor receptor.
Please cite this article as: Hwang S, et al. Tissue expression of tubular injury markers is associated with renal function decline in diabetic nephropathy. Journal of Diabetes and Its Complications (2017), https://doi.org/10.1016/j.jdiacomp.2017.08.009
S. Hwang et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
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Table 3 Correlation of tissue expressions of NGAL, KIM-1, TNFR1, and TNFR2 with clinical and pathologic parameters. Variables
Diabetes duration BMI Hemoglobin A1c eGFR uPCR GFR decline slope Glomerular class IFTA score Inflammatory score Arteriolar hyalinosis score Arteriosclerosis score
r Tubular expression of NGAL
Tubular expression of Glomerular expression KIM-1 of TNFR1
Tubular expression of TNFR1
Glomerular expression of TNFR2
Tubular expression of TNFR2
−0.208 −0.163 0.154 −0.029 0.202 0.601⁎⁎ −0.051 0.391⁎
0.132 −0.361⁎ −0.037 −0.360⁎ 0.596⁎⁎ 0.516⁎⁎ 0.204 0.321 0.131 0.200
0.181 −0.145 −0.110 0.107 0.070 0.188 0.082 0.123 0.175 −0.189
0.205 0.103 −0.190 −0.114 0.165 0.118 −0.221 0.094 0.076 −0.171
−0.092 0.098 −0.050 −0.169 −0.112 −0.296 −0.256 −0.007 0.000 0.038
0.109 0.018 −0.128 −0.280 0.054 −0.009 −0.275 0.174 −0.128 0.055
0.101
0.221
0.069
0.054 0.073 −0.010
−0.178
0.385⁎
Abbreviations: NGAL, neutrophil gelatinase-associated lipocalin; KIM-1, kidney injury molecule-1; TNFR, tumor necrosis factor receptor; BMI, body mass index; eGFR, estimated glomerular filtration rate; uPCR, urinary protein to creatinine ratio; IFTA, interstitial fibrosis and tubular atrophy. ⁎ p-Value b 0.05. ⁎⁎ p-Value b 0.01.
DN histology of humans, and demonstrated that increased NGAL expressions were independently associated with subsequent rapid GFR decline. This finding added strong evidence to the hypothesis that tubular injury plays a critical role in the progression of DKD. Recent studies have demonstrated that KIM-1 is one of the strong prognostic markers in DKD. According to Nowak et al., elevated plasma KIM-1 has been strongly associated with the risk of progressive renal decline in non-proteinuric patients with type 1 diabetes. 16 Sabbisetti et al. found a strong relationship between serum KIM-1 levels and the rate of GFR decline during a 5–15-year follow-up period in 107 patients with diabetes with stage 1–3 CKD at baseline.14 In another study, urinary KIM-1 predicted progression to ESRD in patients with type 1 diabetes with macroalbuminuria, but after adjustment for albuminuria, KIM-1 was no longer a predictor of progression to ESRD. 32 Our results revealed that tubular expressions of KIM-1 were associated with GFR decline slopes. However, the association of KIM-1 expressions and GFR decline slopes was dependent on levels of proteinuria. This finding may reflect a toxic effect of filtered proteins on proximal tubular cells, in which KIM-1 expression is mainly induced by toxic insults. Vaidya et al. showed that urinary excretion of tubular injury marker KIM-1 was significantly associated with time-dependent changes in microalbuminuria in patients with type 1 DM.33
Table 4 Multivariate linear regression analysis of GFR decline slopes with clinical and pathologic parameters, and tubular expressions of NGAL and KIM-1. Variables
Diabetes duration BMI uPCR IFTA score NGAL KIM-1
Model 1
Model 2
Model 3
β
p
β
p
β
P
0.074 −0.366 0.514 0.153
0.584 0.010 0.001 0.252
0.149 −0.313 0.465 0.081 0.290
0.267 0.021 0.001 0.534 0.038
0.062 −0.339 0.467 0.128
0.656 0.022 0.004 0.359
0.112
0.497
Abbreviations: NGAL, neutrophil gelatinase-associated lipocalin; KIM-1, kidney injury molecule-1; GFR, estimated glomerular filtration rate; BMI, body mass index; PCR, protein to creatinine ratio; IFTA, interstitial fibrosis and tubular atrophy. - Model 1: diabetes duration, BMI, and uPCR (clinical parameters with p b 0.2 in univariate analyses) + IFTA score. - Model 2: model 1 + tubular expression of NGAL. - Model 3: model 1 + tubular expression of KIM-1.
A growing body of evidence suggests that the inflammatory process mediates kidney injury in diabetic conditions. 12,34,35 Inflammatory markers such as circulating TNFR1 and TNFR2 have been demonstrated to be strong predictors of DKD progression.11 However, renal expressions of TNFR1 and TNFR2 were not associated with loss of kidney function in the present study. Although both markers showed diverse expressions, no clinical parameters were correlated with tubular or glomerular expressions of TNFR1 and TNFR2. Tubular expression was correlated with glomerular expression in individual patients (data not shown). These findings lead us to consider that systemic sources of TNFR1 and TNFR2 may be involved in the progression of DKD. Several experimental studies have demonstrated that the renin– angiotensin–aldosterone system (RAAS) blockade reduced the tissue expressions of proinflammatory and tubular injury markers. 36,37 Nielsen et al. showed that treatment with RAAS blockade tended to decrease u-NGAL and u-KIM-1 in patients with DN. 28,38 These findings suggested that the RAAS blockade might prevent tubular injury as well as reduce proteinuria in DN. However, there were no significant differences in renal expressions of NGAL and KIM-1 between the treated and untreated groups with ACE-I or ARB in our small retrospective study. Several limitations of this study need to be mentioned. First, the number of subjects was small, so we used GFR decline slopes as outcome parameters. Doubling of the serum creatinine level, a classic endpoint of kidney disease progression, is a late event. GFR change and GFR decline slopes have emerged as alternative endpoints, as their close associations with kidney disease outcomes have been extensively examined. 39,40 Second, we evaluated the tissue expression of biomarkers using only IHC, a semi-quantitative method. Quantitative assays for protein or mRNA expression were not included in the study because of a shortage of kidney tissues. However, IHC studies enabled us to separately assess the glomerular and tubular expression of biomarkers. In addition, IHC staining for each biomarker was simultaneously performed by a single researcher. Third, we enrolled subjects with pathologically proven DN, so our study population was unable to represent the entire spectrum of patients with DKD. The majority of our subjects showed significant decline in kidney function. We believe that rapidly progressive DKD is the area in need of investigation for novel therapeutic interventions. A major strength of our study is that this is the first study examining the expressions of biomarkers in human tissue with pathologically proven DN, and validating correlations between tubular expressions of NGAL and KIM-1, and the progression of DKD.
Please cite this article as: Hwang S, et al. Tissue expression of tubular injury markers is associated with renal function decline in diabetic nephropathy. Journal of Diabetes and Its Complications (2017), https://doi.org/10.1016/j.jdiacomp.2017.08.009
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S. Hwang et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
Table 5 Correlation of tubular expressions with serum and urine levels of NGAL and KIM-1. Variables
Tubular expression of NGAL Tubular expression of KIM-1
a
uNGAL/Cr
b
a
sNGAL
uKIM-1/Cr
β
p
β
p
0.529
0.052
0.247
0.322
b
sKIM-1
β
p
β
p
0.878
b0.001
0.366
0.135
Abbreviations: uNGAL, urine neutrophil gelatinase-associated lipocalin; sNGAL, serum neutrophil gelatinase-associated lipocalin; uKIM-1, urine kidney injury molecule-1; sKIM-1, serum kidney injury molecule-1. a Urine samples were available in 18 subjects. b Serum samples were available in 14 subjects.
In conclusion, the present study demonstrated that tubular expressions of NGAL and KIM-1, tubular injury markers, were closely associated with GFR decline slopes in patients with pathologically proven DN. This finding suggests that tubular injury has a key role in the pathogenesis of DKD in patients at a high risk for kidney disease progression. Further studies are warranted to determine whether tubular injury can be a therapeutic target in DKD. Acknowledgements We thank Kyung Yi Choi, Ji Woo Kim, and Woo Hyun Lee of the Samsung Biomedical Research Institute for providing technical assistance. Grants This study was supported by a grant from the Samsung Biomedical Research Institute Grant (Grant No: OTC 1601971). References 1. Gregg EW, Li Y, Wang J, Burrows NR, Ali MK, Rolka D, et al. Changes in diabetes-related complications in the United States, 1990–2010. N Engl J Med. 2014;370:1514-23. 2. Pavkov ME, Knowler WC, Lemley KV, Mason CC, Myers BD, Nelson RG. Early renal function decline in type 2 diabetes. Clin J Am Soc Nephrol. 2012;7:78-84. 3. Berhane AM, Weil EJ, Knowler WC, Nelson RG, Hanson RL. Albuminuria and estimated glomerular filtration rate as predictors of diabetic end-stage renal disease and death. Clin J Am Soc Nephrol. 2011;6:2444-51. 4. Keane WF, Brenner BM, de Zeeuw D, Grunfeld JP, McGill J, Mitch WE, et al. The risk of developing end-stage renal disease in patients with type 2 diabetes and nephropathy: the RENAAL study. Kidney Int. 2003;63:1499-507. 5. Yokoyama H, Kanno S, Takahashi S, Yamada D, Itoh H, Saito K, et al. Determinants of decline in glomerular filtration rate in nonproteinuric subjects with or without diabetes and hypertension. Clin J Am Soc Nephrol. 2009;4:1432-40. 6. Lewis EJ, Hunsicker LG, Clarke WR, Berl T, Pohl MA, Lewis JB, et al. Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes. N Engl J Med. 2001;345:851-60. 7. Hou FF, Zhang X, Zhang GH, Xie D, Chen PY, Zhang WR, et al. Efficacy and safety of benazepril for advanced chronic renal insufficiency. N Engl J Med. 2006;354: 131-40. 8. Hovind P, Rossing P, Tarnow L, Smidt UM, Parving HH. Progression of diabetic nephropathy. Kidney Int. 2001;59:702-9. 9. Thakar CV, Christianson A, Himmelfarb J, Leonard AC. Acute kidney injury episodes and chronic kidney disease risk in diabetes mellitus. Clin J Am Soc Nephrol. 2011;6:2567-72. 10. Perkins BA, Ficociello LH, Ostrander BE, Silva KH, Weinberg J, Warram JH, et al. Microalbuminuria and the risk for early progressive renal function decline in type 1 diabetes. J Am Soc Nephrol. 2007;18:1353-61. 11. Niewczas MA, Gohda T, Skupien J, Smiles AM, Walker WH, Rosetti F, et al. Circulating TNF receptors 1 and 2 predict ESRD in type 2 diabetes. J Am Soc Nephrol. 2012;23:507-15. 12. Sun L, Kanwar YS. Relevance of TNF-alpha in the context of other inflammatory cytokines in the progression of diabetic nephropathy. Kidney Int. 2015;88:662-5.
13. Kim SS, Song SH, Kim IJ, Yang JY, Lee JG, Kwak IS, et al. Clinical implication of urinary tubular markers in the early stage of nephropathy with type 2 diabetic patients. Diabetes Res Clin Pract. 2012;97:251-7. 14. Sabbisetti VS, Waikar SS, Antoine DJ, Smiles A, Wang C, Ravisankar A, et al. Blood kidney injury molecule-1 is a biomarker of acute and chronic kidney injury and predicts progression to ESRD in type I diabetes. J Am Soc Nephrol. 2014;25: 2177-86. 15. de Carvalho JA, Tatsch E, Hausen BS, Bollick YS, Moretto MB, Duarte T, et al. Urinary kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin as indicators of tubular damage in normoalbuminuric patients with type 2 diabetes. Clin Biochem. 2016;49:232-6. 16. Nowak N, Skupien J, Niewczas MA, Yamanouchi M, Major M, Croall S, et al. Increased plasma kidney injury molecule-1 suggests early progressive renal decline in non-proteinuric patients with type 1 diabetes. Kidney Int. 2016;89:459-67. 17. Gilbert RE, Cooper ME. The tubulointerstitium in progressive diabetic kidney disease: More than an aftermath of glomerular injury? Kidney Int. 1999;56:1627-37. 18. Magri CJ, Fava S. The role of tubular injury in diabetic nephropathy. Eur J Intern Med. 2009;20:551-5. 19. Thomas MC, Burns WC, Cooper ME. Tubular changes in early diabetic nephropathy. Adv Chronic Kidney Dis. 2005;12:177-86. 20. Levey AS, Stevens LA, Schmid CH, Zhang YL, Castro 3rd AF, Feldman HI, et al. A new equation to estimate glomerular filtration rate. Ann Intern Med. 2009;150: 604-12. 21. Tervaert TW, Mooyaart AL, Amann K, Cohen AH, Cook HT, Drachenberg CB, et al. Pathologic classification of diabetic nephropathy. J Am Soc Nephrol. 2010;21: 556-63. 22. An Y, Xu F, Le W, Ge Y, Zhou M, Chen H, et al. Renal histologic changes and the outcome in patients with diabetic nephropathy. Nephrol Dial Transplant. 2015;30:257-66. 23. Mise K, Hoshino J, Ueno T, Hazue R, Sumida K, Hiramatsu R, et al. Clinical and pathological predictors of estimated GFR decline in patients with type 2 diabetes and overt proteinuric diabetic nephropathy. Diabetes Metab Res Rev. 2015;31:572-81. 24. Abbate M, Zoja C, Remuzzi G. How does proteinuria cause progressive renal damage? J Am Soc Nephrol. 2006;17:2974-84. 25. Brito PL, Fioretto P, Drummond K, Kim Y, Steffes MW, Basgen JM, et al. Proximal tubular basement membrane width in insulin-dependent diabetes mellitus. Kidney Int. 1998;53:754-61. 26. Nakhoul F, Nakhoul N, Asleh R, Miller-Lotan R, Levy AP. Is the Hp 2-2 diabetic mouse model a good model to study diabetic nephropathy? Diabetes Res Clin Pract. 2013;100:289-97. 27. Nauta FL, Boertien WE, Bakker SJ, van Goor H, van Oeveren W, de Jong PE, et al. Glomerular and tubular damage markers are elevated in patients with diabetes. Diabetes Care. 2011;34:975-81. 28. Nielsen SE, Schjoedt KJ, Astrup AS, Tarnow L, Lajer M, Hansen PR, et al. Neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1 (KIM1) in patients with diabetic nephropathy: a cross-sectional study and the effects of lisinopril. Diabet Med. 2010;27:1144-50. 29. Bolignano D, Lacquaniti A, Coppolino G, Donato V, Fazio MR, Nicocia G, et al. Neutrophil gelatinase-associated lipocalin as an early biomarker of nephropathy in diabetic patients. Kidney Blood Press Res. 2009;32:91-8. 30. Yang YH, He XJ, Chen SR, Wang L, Li EM, Xu LY. Changes of serum and urine neutrophil gelatinase-associated lipocalin in type-2 diabetic patients with nephropathy: One year observational follow-up study. Endocrine. 2009;36:45-51. 31. Devarajan P. The use of targeted biomarkers for chronic kidney disease. Adv Chronic Kidney Dis. 2010;17:469-79. 32. Panduru NM, Sandholm N, Forsblom C, Saraheimo M, Dahlstrom EH, Thorn LM, et al. Kidney injury molecule-1 and the loss of kidney function in diabetic nephropathy: a likely causal link in patients with type 1 diabetes. Diabetes Care. 2015;38:1130-7. 33. Vaidya VS, Niewczas MA, Ficociello LH, Johnson AC, Collings FB, Warram JH, et al. Regression of microalbuminuria in type 1 diabetes is associated with lower levels of urinary tubular injury biomarkers, kidney injury molecule-1, and N-acetyl-beta-D-glucosaminidase. Kidney Int. 2011;79:464-70. 34. Navarro-Gonzalez JF, Mora-Fernandez C. The role of inflammatory cytokines in diabetic nephropathy. J Am Soc Nephrol. 2008;19:433-42. 35. Al-Lamki RS, Mayadas TN. TNF receptors: signaling pathways and contribution to renal dysfunction. Kidney Int. 2015;87:281-96. 36. Kelly DJ, Cox AJ, Tolcos M, Cooper ME, Wilkinson-Berka JL, Gilbert RE. Attenuation of tubular apoptosis by blockade of the renin-angiotensin system in diabetic Ren-2 rats. Kidney Int. 2002;61:31-9. 37. Giani JF, Burghi V, Veiras LC, Tomat A, Munoz MC, Cao G, et al. Angiotensin-(1-7) attenuates diabetic nephropathy in Zucker diabetic fatty rats. Am J Physiol Renal Physiol. 2012;302:F1606-15. 38. Nielsen SE, Rossing K, Hess G, Zdunek D, Jensen BR, Parving HH, et al. The effect of RAAS blockade on markers of renal tubular damage in diabetic nephropathy: u-NGAL, u-KIM1 and u-LFABP. Scand J Clin Lab Invest. 2012;72:137-42. 39. Xie Y, Bowe B, Xian H, Balasubramanian S, Al-Aly Z. Estimated GFR trajectories of people entering CKD stage 4 and subsequent kidney disease outcomes and mortality. Am J Kidney Dis. 2016;68:219-28. 40. Skupien J, Warram JH, Smiles AM, Niewczas MA, Gohda T, Pezzolesi MG, et al. The early decline in renal function in patients with type 1 diabetes and proteinuria predicts the risk of end-stage renal disease. Kidney Int. 2012;82:589-97.
Please cite this article as: Hwang S, et al. Tissue expression of tubular injury markers is associated with renal function decline in diabetic nephropathy. Journal of Diabetes and Its Complications (2017), https://doi.org/10.1016/j.jdiacomp.2017.08.009
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Journal of Diabetes and Its Complications journal homepage: www.jdcjournal.com
Tissue expression of tubular injury markers is associated with renal function decline in diabetic nephropathy Subin Hwang a, Jeeeun Park a, Jinhae Kim a, Hye Ryoun Jang a, Ghee Young Kwon b, Wooseong Huh a, Yoon-Goo Kim a, Dae Joong Kim a, Ha Young Oh a, Jung Eun Lee a,⁎ a b
Nephrology Division, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 10 May 2017 Received in revised form 28 July 2017 Accepted 20 August 2017 Available online xxxx Keywords: Diabetic nephropathy Diabetic kidney disease Neutrophil gelatinase-associated lipocalin Kidney injury molecule-1 Immunohistochemistry
a b s t r a c t Aims: The pathogenesis of diabetic kidney disease (DKD) is complex and multifactorial; increasing evidence suggests that tubular injury and inflammatory process are involved in disease progression. We investigated the potential association of renal expression of tubular injury markers, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and inflammatory markers, tumor necrosis factor receptor (TNFR) 1 and 2 with renal progression in pathologically proven diabetic nephropathy (DN). Methods: We identified 122 patients with confirmed DN. After excluding patients with other coexisting renal disease or estimated glomerular filtration rate (eGFR) b 30 mL/min/1.73 m2, 35 patients were included. Annual decline of (GFR decline slope) was calculated using linear regression analysis. Tissue tubular and glomerular expressions of NGAL, KIM-1, TNFR1, and TNFR2 were assessed using immunohistochemistry. Results: Median baseline urinary protein to creatinine ratio (uPCR) was 6.76 (2.18–7.61) mg/mg Cr, median baseline eGFR was 50 (43–66) mL/min per 1.73 m2, and median GFR decline slope was 15.6 (4.4–35.1) mL/ min per 1.73 m2 per year. Positive correlations were observed between tubular expressions of NGAL and KIM-1, and GFR decline slopes (r = 0.601, p b 0.001; r = 0.516, p = 0.001, respectively), and between tubular expressions of KIM-1 and uPCR (r = 0.596, p b 0.001), and between NGAL and interstitial fibrosis and tubular atrophy (IFTA) score (r = 0.391, p = 0.024). No correlations were found between glomerular or tubular expressions of TNFRs, and clinical parameters including GFR decline slopes. On multivariate analysis, the association between tubular expressions of KIM-1 and GFR decline slopes was dependent on uPCR. Tubular expressions of NGAL were independently associated with GFR decline slopes, with an adjusted coefficient factor of 0.290 (95% confidence interval, 0.009–0.202, p = 0.038). Conclusions: These findings suggest that tubular injury plays a key role in the pathogenesis of DKD in high-risk patients. Further studies are warranted to determine whether tubular injury could be a therapeutic target in DKD. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Despite major implementation of renoprotective treatment in recent decades, diabetic kidney disease (DKD) continues to rank as the leading cause of end-stage renal disease (ESRD). Paradoxically, improvements in cardiovascular outcomes in patients with diabetes have allowed ample time for the development of kidney dysfunction and ESRD. 1 Therefore, prevention and delay of DKD progression is becoming increasingly important to reduce public health burdens. Conflict of Interest Statement: All authors declare that they have no conflict of interest. ⁎ Corresponding author at: Division of Nephrology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, 06351, Seoul, Republic of Korea. E-mail address: [email protected] (J.E. Lee).
Susceptibilities to DKD and outcomes are highly variable. The amount of proteinuria, elevated blood pressure (BP), decreased kidney function, hyperglycemia, and episodes of acute kidney injury (AKI) have been identified as risk factors for the progression of DKD. 2–10 Classically, glomeruli have been considered the primary injury site for diabetic nephropathy (DN), and albuminuria, which reflects glomerular damage, has been a key prognostic marker. Recently, several biomarkers related to inflammation or tubular injury have been identified as potent predictors of renal outcome. Circulating tumor necrosis factor receptor (TNFR) 1 and 2 have been reported to be strongly associated with subsequent progression to ESRD in patients with diabetes.11,12 Furthermore, urinary and plasma levels of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) may function as early markers of DN. 13–16
https://doi.org/10.1016/j.jdiacomp.2017.08.009 1056-8727/© 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Hwang S, et al. Tissue expression of tubular injury markers is associated with renal function decline in diabetic nephropathy. Journal of Diabetes and Its Complications (2017), https://doi.org/10.1016/j.jdiacomp.2017.08.009
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Accumulating evidence indicates that the inflammatory process and tubular damage are early events in the course of DN and are not merely secondary to glomerular damage.17–19 In the present study, we investigated whether renal expression of NGAL, KIM-1, TNFR1, and TNFR2 predict subsequent decline of kidney function in subjects with pathologically proven DN. 2. Materials and methods 2.1. Population We identified 122 patients with diabetes mellitus (DM) who underwent renal biopsy and were confirmed to have DN between January 2000 and December 2014 at a 2000-bed tertiary referral center in Seoul, Korea. We excluded subjects with other coexisting renal disease based on pathologic findings (other types of glomerulonephritis, acute tubular necrosis, or acute interstitial nephritis) (n = 24), those with estimated GFR (eGFR) b30 mL/min/1.73 m 2 at the time of biopsy (n = 54), those who received treatment with immunosuppressants (n = 2), and those who underwent follow-up for b 6 months or b3 visits after renal biopsy (n = 7). Finally, 35 subjects were included in the analyses. All included subjects had provided written informed consent at the time of biopsy for the collection and use of their remnant biopsy specimens, and serum and urinary samples for research regarding biomarkers. This study was approved by the institutional review board of Samsung Medical Center. As this study was retrospective and the subjects were de-identified, the institutional review board waived the need for additional written consent from the subjects. 2.2. Clinical and laboratory data Data regarding age, sex, duration and type of diabetes, presence of diabetic retinopathy, anti-hypertensive treatment including angiotensin-converting enzyme inhibitor (ACE-I) or angiotensin receptor blocker (ARB), body mass index (BMI), smoking status, and systolic and diastolic (BP) at the time of renal biopsy were obtained from electronic medical records of the patients. Hemoglobin A1c (HbA1c) levels at the time of renal biopsy were also obtained. The average of the urinary protein to creatinine ratio (uPCR) in spot urine samples obtained during the 6 months after renal biopsy was used as the baseline time-averaged uPCR. The primary outcome was the GFR decline slope (in mL/min/1.73 m 2 per year). To estimate the GFR decline slopes for each patient, we collected all serum creatinine measurements from 3 months before renal biopsy to the day of the last follow-up. We then calculated eGFR using the Chronic Kidney Disease Epidemiology Collaboration formula. 20 Next, a model for time versus eGFR was created using a linear regression analysis, and the absolute values of the slope of the regression line were regarded as GFR decline slopes over time, with a positive value representing a decreasing trajectory of GFR. 2.3. Renal biopsy and pathologic classification Kidney histology was retrospectively reviewed. The tissues had been obtained by needle biopsy, and the specimens had been processed for light microscopy and immunofluorescence, as well as for electron microscopy. Classification of DN and histological scoring were performed according to the criteria of Mooyaar et al. 21 Renal pathological findings were also included as categorical covariates: glomerular class (I, IIa, IIb, III, IV); interstitial fibrosis and tubular atrophy (IFTA) score (0, 1, 2, 3); interstitial inflammation score (0, 1, 2); arteriolar hyalinosis score (0, 1, 2); and arteriosclerosis score (0, 1, 2).
2.4. Immunohistochemical (IHC) study Serial sections of formalin-fixed paraffin-embedded kidney tissues were prepared and placed on a microscope slide. After deparaffinization and rehydration, tissue sections were washed, followed by blocking of endogenous peroxidase through incubation with Dako REAL Peroxidase Blocking solution (DAKO, Seoul, South Korea) for 30 min at room temperature. In addition, tissues prepared for NGAL were incubated with avidin and biotin for 15 min. Then, all tissues were treated with Dako Protein Block Serum-Free agent (DAKO) overnight at 4 °C to prevent background staining. Subsequently, tissue sections were incubated in primary antibodies KIM1 (Human TIM-1/KIM-1/HAVCR Ab; R&D Systems, Boston, MA, USA; 1:300), NGAL (Human Lipocalin-2/NGAL Antibody; R&D Systems; 1:100), and TNFR1 and TNFR2 (Anti-TNF Receptor I Ab and II Ab; Abcam, Cambridge, UK; 1:500 and 1:50, respectively) for 1 h at room temperature. Thereafter, the tissue sections were incubated with a secondary antibody (EnVision for KIM-1, TNFR1, and TNFR2; rabbit polyclonal antibody to rat I immunoglobulin G-biotin for NGAL) for 30 min. Then, streptavidin horseradish peroxidase (HRP) for tissues that reacted with NGAL antibody was added, along with 3,3′ diaminobenzidine (DAB) chromogenic for visualization in a color reaction, followed by hematoxylin dye. Brown staining showed positive results. Staining levels were scored as follows. The IHC staining was evaluated by the percentage and intensity of positive tubular or glomerular cells. The percentage of positive cells was recorded as 0 (0–10%), 1 (11–25%), 2 (26–50%), 3 (51%–75%), or 4 (N 75%). The staining intensity of positive cells was recorded as 0 (negative), 1 (weakly positive), 2 (moderately positive), or 3 (strongly positive). The total score (0−12) was calculated by multiplying the two parameters. Positive immunoreactivity for NGAL and KIM-1 was predominantly detected in renal tubules, while immunoreactivity for TNFR1 and TNFR2 was detected in both tubules and glomeruli. Therefore, the glomerular and tubular expressions of TNFRs were separately assessed. 2.5. Serum and urine NGAL and KIM-1 measurements Serum NGAL (sNGAL), serum KIM-1 (sKIM-1), urinary creatinine, urinary NGAL (uNGAL), and urinary KIM-1 (uKIM-1) concentrations were measured using enzyme-linked immunosorbent assay (ELISA) kits (NGAL: Lipocalin-2/NGAL, R&D Systems; KIM-1: TIM-1/KIM-1/ HAVCR, R&D Systems), according to the manufacturer's instructions. Measurements were performed in duplicate and triplicate for serum and urine, respectively. 2.6. Statistical analyses Data were expressed as percentages for categorical variables and as medians (interquartile ranges, IQRs) for continuous variables. As some variables (GFR decline slope, HbA1c, and uPCR) showed skewed distributions, log transformation was carried out before performing correlation and regression analysis. The Mann–Whitney rank sum test was used for comparisons of continuous variables, and Fisher's exact test was used for categorical variables. Correlations among continuous variables were assessed using the Spearman rank correlation coefficient. To investigate the variables that could predict rapid progression of DN, multivariate linear regression analysis was conducted to explore the association of GFR decline slopes with baseline clinical and histopathological variables, and tubular expression of NGAL and KIM-1 following univariate linear regression with p b 0.2, and the significant explanatory parameters were chosen in an enter stepwise manner. Results were expressed as the regression coefficient (β).
Please cite this article as: Hwang S, et al. Tissue expression of tubular injury markers is associated with renal function decline in diabetic nephropathy. Journal of Diabetes and Its Complications (2017), https://doi.org/10.1016/j.jdiacomp.2017.08.009
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Statistical significance was defined as p b 0.05. All statistical analyses were performed using SPSS for Windows, version 23.0 (IBM Corp., Chicago, IL, USA).
3. Results Of the 35 subjects, 28 were men (80%). At the time of biopsy, the median age of the subjects was 50 (43–59) years (Table 1). The duration of DM was 105–14 years, and 83% of the subjects had diabetic retinopathy. The median follow-up duration after biopsy was 24.2 (13.5–51.2) months. The median time-averaged uPCR was 6.76 (2.18–7.61) mg/mg Cr and the median baseline eGFR was 50 (43–66) mL/min/1.73 m 2. Based on the glomerular classification, 1 (3%) subject was in class I, 2 subjects (6%) in class IIa, 4 (11%) in class IIb, 24 (69%) in class III, and 4 (11%) in class IV. IFTA scores of 0, 1, 2 and 3 were observed in 2 (6%), 20 (57%), 12 (34%), and 1 (3%) subjects, respectively. Interstitial inflammation of scores 0, 1, and 2 was observed in 2 (6%), 30 (86%), and 3 (8%) subjects, respectively. Arteriolar hyalinosis was absent in 5 subjects (14%). More than one area of arteriolar hyalinosis (scored as 2) was found in 28 subjects (80%). In 2 subjects, large vessels were absent in renal biopsies. Among the remaining 33 subjects, 6 (18%) had no intimal thickening (scored as 0) and 14 (42%) had severe arteriosclerosis (scored as 2). The median GFR decline slope was 15.6 (4.4–35.1) mL/min/1.73 m 2 per year. Evaluation of the correlation of pathologic classification with clinical parameters (Table 2) showed that IFTA scores were positively correlated with GFR decline slopes (r = 0.415, p = 0.024) and
Table 1 Baseline clinical and pathologic characteristics. Variables
n = 35
Age (years) Female, n, (%) Follow-up duration (months) Diabetes duration (years) Diabetes type, n (%) Type 1/type 2 Diabetic retinopathy, n (%) Body mass index (kg/m2) Smoking status, n (%) Never/ex-/current smoker Systolic BP (mm Hg) Diastolic BP (mm Hg) ACE-I or ARB treatment, n (%) Hemoglobin A1c (%) uPCR (mg/mg Cr) CKD stage, n (%) 1/2/3A/3B eGFR (mL/min/1.73 m2) GFR decline slope (mL/min/1.73 m2 per year) Glomerular class, n (%) I/IIa/IIb/III/IV IFTA, n (%) 0/1/2/3 Interstitial inflammation score, n (%) 0/1/2 Arteriolar hyalinosis score, n, (%) 0/1/2 Arteriosclerosis scorea, n (%) 0/1/2
50 (43–59) 7 (20%) 24.2 (13.5–51.2) 10 (5–14) 2 (6%)/33 (94%) 29 (83%) 23.6 (22.4–25.5) 21 (60%)/5 (14%)/9 (26%) 139 (132–150) 80 (73–89) 28 (80%) 7.3 (6.0–8.2) 6.76 (2.18–7.61) 4 (11%)/7 (20%)/12 (34%)/12 (34%) 50 (43–66) 15.6 (4.4–35.1)
1 (3%)/2 (6%)/4 (11%)/24 (69%)/4 (11%) 2 (6%)/20 (57%)/12 (34%)/1 (3%) 2 (6%)/30 (86%)/3 (8%) 5 (14%)/2 (6%)/28 (80%) 6 (18%)/13 (39%)/14 (42%)
Continuous variables were expressed as median (interquartile range). Abbreviations: BP, blood pressure; ACE-I, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; uPCR, urinary protein to creatinine ratio; CKD, chronic kidney disease; eGFR, estimated glomerular filtration rate; IFTA, interstitial fibrosis and tubular atrophy. a Arteriosclerosis score was missing for two subjects because their renal biopsy specimen did not include any arteries.
3
arteriolar hyalinosis scores were correlated with uPCR (r = 0.399, p = 0.018). Additionally, negative and positive correlations were observed, respectively, between interstitial inflammation scores and BMI (r = −0.336, p = 0.048), and between arteriosclerosis scores and diabetes duration (r = 0.359, p = 0.040). Tissue expression of NGAL, KIM-1, TNFR1, and TNFR2 was examined in each patient (Fig. 1). The correlations between tissue expressions of NGAL, KIM-1, TNFR1, and TNFR2, and clinical and pathologic parameters are presented in Table 3. There were positive correlations between tubular expressions of NGAL and KIM-1, and GFR decline slopes (r = 0.601, p b 0.001; r = 0.516, p = 0.001, respectively). Moreover, the expressions of KIM-1 and uPCR (r = 0.596, p b 0.001) and the expressions of NGAL and IFTA scores (r = 0.391, p = 0.024) showed positive correlations. However, no correlations were observed between glomerular or tubular expressions of TNFRs and clinical parameters including GFR decline slopes. When examining the renal expressions of NGAL and KIM-1 according to treatment status with ACE-I or ARB, the median score of tubular expressions of NGAL was 5 4–6 in the group treated with ACE-I or ARB and 3 3–5 in the group not treated with ACE-I or ARB. The median score of tubular expressions of KIM-1 was 6 (3–7.8) in the group treated with ACE-I or ARB, and it was 5 4–8 in the others. There were no significant differences between the groups (p = 0.444 and p = 0.806, respectively). To identify parameters to predict the GFR decline slope, linear regression analyses were conducted. Among the clinical parameters, diabetes duration, BMI, and uPCR were associated with GFR decline slopes (β = 0.026, p = 0.184 for diabetes duration; β = 0.926, p b 0.001 for uPCR; and β = −0.079, p = 0.012 for BMI, respectively) in univariate analyses. Multivariate analysis revealed that the association between tubular expressions of KIM-1 and GFR decline slopes was dependent on uPCR. In contrast, tubular expressions of NGAL demonstrated an independent association with GFR decline slopes, with an adjusted coefficient factor of 0.290 (95% confidence interval [CI], 0.009–0.202, p = 0.038) (Table 4). For analyses of NGAL and KIM-1 concentrations, serum samples were available in 14 subjects and urine samples were available in 18 subjects. Tubular expressions of KIM-1 were not correlated with sKIM-1 (r = 0.366, p = 0.135), but those were highly correlated with uKIM-1 (r = 0.878, p b 0.001). Tubular expressions of NGAL showed a positive correlation with uNGAL (r = 0.529, p = 0.052) (Table 5). 4. Discussion In the present study, we evaluated the relationship between the tissue expressions of NGAL, KIM-1, TNFR1 and TNFR2, clinical and histopathologic parameters, and renal progression in subjects with pathologically proven DN. Proteinuria is a major clinical determinant of subsequent GFR decline slopes, and tubulointerstitial change is the most important histologic finding associated with the progression of DN. Renal expressions of TNFR1 and TNFR2 were not associated with proteinuria and baseline GFR or with GFR decline slope in the current study. Meanwhile, increased tubular expressions of NGAL and KIM-1 were associated with subsequent GFR decline. Moreover, the association of tubular expressions of NGAL and GFR decline slopes was independent of proteinuria and tubulointerstitial change. Considering that this study included patients with rapidly progressive DN, these findings suggest that tubular injury plays a key role in the pathogenesis of DN in patients at high risk. KIM-1 and NGAL are well-known tubular injury markers. It is now increasingly recognized that tubular injury has an important role in the pathogenesis and progression of DN, and the severity of tubulointerstitial lesions has a significant impact on renal outcome in DN. 22,23 The mechanisms that underlie tubular injury are
Please cite this article as: Hwang S, et al. Tissue expression of tubular injury markers is associated with renal function decline in diabetic nephropathy. Journal of Diabetes and Its Complications (2017), https://doi.org/10.1016/j.jdiacomp.2017.08.009
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Table 2 Correlation of pathologic classification with clinical parameters. Variables
Age Diabetes duration BMI Smoking status Hemoglobin A1c eGFR uPCR GFR decline slope
r Glomerular class
IFTA score
Interstitial inflammation score
Arteriolar hyalinosis score
Arteriosclerosis score
0.012 0.009 −0.147 −0.352⁎ 0.180 −0.008 −0.005 0.025
−0.291 −0.017 −0.029 −0.033 0.103 −0.094 0.244 0.415⁎
−0.007 −0.020 −0.336⁎
−0.203 −0.016 0.023 0.224 0.321 0.166 0.399⁎ 0.293
0.313 0.359⁎ −0.002 0.192 0.146 −0.073 0.056 0.078
−0.289 0.073 −0.033 0.203 0.034
Abbreviations: IFTA, interstitial fibrosis and tubular atrophy; BMI, body mass index; BP, blood pressure; eGFR, estimated glomerular filtration rate; uPCR, urinary protein to creatinine ratio. ⁎ p-Value b 0.05.
complex. 18 Tubular epithelial cells are persistently exposed to hyperglycemic conditions by tubular reabsorption of glucose in patients with diabetes.19 In addition, the local renin–angiotensin system and chronic hypoxia contribute to progressive tubular injury. Furthermore, albuminuria may be directly harmful to renal tubular cells, leading to tubular inflammation and fibrosis.24 The proximal tubule, in particular, is more vulnerable to these metabolic and hemodynamic factors. Brito et al. 25 have shown that the proximal tubular basement membrane is already thickened in normoalbuminuric patients with diabetes. In addition, a few studies have demonstrated that excessive iron deposit can lead to increased oxidative stress injury in proximal tubule, leading to cell damage. 26 The present study is, to the best of our knowledge, the first to demonstrate, using human histology, that increased tissue expressions of tubular injury markers are associated with progressive loss of kidney function. Further studies
are needed to evaluate whether modulation of tubular injury could be a target of new therapeutic intervention in DN. Serum and urinary NGAL levels have been studied as potential biomarkers of DKD, demonstrating correlations with GFR and/or albuminuria.27–30 However, the prognostic power of these markers to predict the progression of DKD has not exceeded the power of clinical parameters. 31 One limitation of serum or urine NGAL levels as biomarkers is that the NGAL protein is not specific to kidney injury. In other words, the protein can be released into the circulation from any organs with inflammation. Furthermore, serum NGAL can be filtered in the glomerulus, so serum levels are highly dependent on the current glomerular filtration rate, even in circumstances other than those of kidney injury. Thus, serum and urinary levels of NGAL may not directly reflect intrarenal NGAL expression in patients with CKD. The present study evaluated the tubular expression of NGAL in
Fig. 1. Tissue expression of NGAL, KIM-1, and TNFR1 and TNFR2 in kidneys with diabetic nephropathy. Representative images of immunohistochemistry (IHC)-stained section of the kidney with diabetic nephropathy (original magnification, ×200). IHC sections for NGAL (A–D), KIM-1 (E–H), TNFR1 (I–K), and TNFR2 (L–O). Percentage of positive cells was recorded as 0 (0–10%), 1 (11–25%), 2 (26–50%), 3 (51–75%), or 4 (N75%), and staining intensity was recorded as 0 (negative), 1 (weakly positive), 2 (moderately positive), or 3 (strongly positive). Total score (0–12) was calculated by multiplying the two parameters. There was no specimen compatible with “negative” for TNFR1. Abbreviations: NGAL, neutrophil gelatinase-associated lipocalin; KIM-1, kidney injury molecule-1; TNFR, tumor necrosis factor receptor.
Please cite this article as: Hwang S, et al. Tissue expression of tubular injury markers is associated with renal function decline in diabetic nephropathy. Journal of Diabetes and Its Complications (2017), https://doi.org/10.1016/j.jdiacomp.2017.08.009
S. Hwang et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
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Table 3 Correlation of tissue expressions of NGAL, KIM-1, TNFR1, and TNFR2 with clinical and pathologic parameters. Variables
Diabetes duration BMI Hemoglobin A1c eGFR uPCR GFR decline slope Glomerular class IFTA score Inflammatory score Arteriolar hyalinosis score Arteriosclerosis score
r Tubular expression of NGAL
Tubular expression of Glomerular expression KIM-1 of TNFR1
Tubular expression of TNFR1
Glomerular expression of TNFR2
Tubular expression of TNFR2
−0.208 −0.163 0.154 −0.029 0.202 0.601⁎⁎ −0.051 0.391⁎
0.132 −0.361⁎ −0.037 −0.360⁎ 0.596⁎⁎ 0.516⁎⁎ 0.204 0.321 0.131 0.200
0.181 −0.145 −0.110 0.107 0.070 0.188 0.082 0.123 0.175 −0.189
0.205 0.103 −0.190 −0.114 0.165 0.118 −0.221 0.094 0.076 −0.171
−0.092 0.098 −0.050 −0.169 −0.112 −0.296 −0.256 −0.007 0.000 0.038
0.109 0.018 −0.128 −0.280 0.054 −0.009 −0.275 0.174 −0.128 0.055
0.101
0.221
0.069
0.054 0.073 −0.010
−0.178
0.385⁎
Abbreviations: NGAL, neutrophil gelatinase-associated lipocalin; KIM-1, kidney injury molecule-1; TNFR, tumor necrosis factor receptor; BMI, body mass index; eGFR, estimated glomerular filtration rate; uPCR, urinary protein to creatinine ratio; IFTA, interstitial fibrosis and tubular atrophy. ⁎ p-Value b 0.05. ⁎⁎ p-Value b 0.01.
DN histology of humans, and demonstrated that increased NGAL expressions were independently associated with subsequent rapid GFR decline. This finding added strong evidence to the hypothesis that tubular injury plays a critical role in the progression of DKD. Recent studies have demonstrated that KIM-1 is one of the strong prognostic markers in DKD. According to Nowak et al., elevated plasma KIM-1 has been strongly associated with the risk of progressive renal decline in non-proteinuric patients with type 1 diabetes. 16 Sabbisetti et al. found a strong relationship between serum KIM-1 levels and the rate of GFR decline during a 5–15-year follow-up period in 107 patients with diabetes with stage 1–3 CKD at baseline.14 In another study, urinary KIM-1 predicted progression to ESRD in patients with type 1 diabetes with macroalbuminuria, but after adjustment for albuminuria, KIM-1 was no longer a predictor of progression to ESRD. 32 Our results revealed that tubular expressions of KIM-1 were associated with GFR decline slopes. However, the association of KIM-1 expressions and GFR decline slopes was dependent on levels of proteinuria. This finding may reflect a toxic effect of filtered proteins on proximal tubular cells, in which KIM-1 expression is mainly induced by toxic insults. Vaidya et al. showed that urinary excretion of tubular injury marker KIM-1 was significantly associated with time-dependent changes in microalbuminuria in patients with type 1 DM.33
Table 4 Multivariate linear regression analysis of GFR decline slopes with clinical and pathologic parameters, and tubular expressions of NGAL and KIM-1. Variables
Diabetes duration BMI uPCR IFTA score NGAL KIM-1
Model 1
Model 2
Model 3
β
p
β
p
β
P
0.074 −0.366 0.514 0.153
0.584 0.010 0.001 0.252
0.149 −0.313 0.465 0.081 0.290
0.267 0.021 0.001 0.534 0.038
0.062 −0.339 0.467 0.128
0.656 0.022 0.004 0.359
0.112
0.497
Abbreviations: NGAL, neutrophil gelatinase-associated lipocalin; KIM-1, kidney injury molecule-1; GFR, estimated glomerular filtration rate; BMI, body mass index; PCR, protein to creatinine ratio; IFTA, interstitial fibrosis and tubular atrophy. - Model 1: diabetes duration, BMI, and uPCR (clinical parameters with p b 0.2 in univariate analyses) + IFTA score. - Model 2: model 1 + tubular expression of NGAL. - Model 3: model 1 + tubular expression of KIM-1.
A growing body of evidence suggests that the inflammatory process mediates kidney injury in diabetic conditions. 12,34,35 Inflammatory markers such as circulating TNFR1 and TNFR2 have been demonstrated to be strong predictors of DKD progression.11 However, renal expressions of TNFR1 and TNFR2 were not associated with loss of kidney function in the present study. Although both markers showed diverse expressions, no clinical parameters were correlated with tubular or glomerular expressions of TNFR1 and TNFR2. Tubular expression was correlated with glomerular expression in individual patients (data not shown). These findings lead us to consider that systemic sources of TNFR1 and TNFR2 may be involved in the progression of DKD. Several experimental studies have demonstrated that the renin– angiotensin–aldosterone system (RAAS) blockade reduced the tissue expressions of proinflammatory and tubular injury markers. 36,37 Nielsen et al. showed that treatment with RAAS blockade tended to decrease u-NGAL and u-KIM-1 in patients with DN. 28,38 These findings suggested that the RAAS blockade might prevent tubular injury as well as reduce proteinuria in DN. However, there were no significant differences in renal expressions of NGAL and KIM-1 between the treated and untreated groups with ACE-I or ARB in our small retrospective study. Several limitations of this study need to be mentioned. First, the number of subjects was small, so we used GFR decline slopes as outcome parameters. Doubling of the serum creatinine level, a classic endpoint of kidney disease progression, is a late event. GFR change and GFR decline slopes have emerged as alternative endpoints, as their close associations with kidney disease outcomes have been extensively examined. 39,40 Second, we evaluated the tissue expression of biomarkers using only IHC, a semi-quantitative method. Quantitative assays for protein or mRNA expression were not included in the study because of a shortage of kidney tissues. However, IHC studies enabled us to separately assess the glomerular and tubular expression of biomarkers. In addition, IHC staining for each biomarker was simultaneously performed by a single researcher. Third, we enrolled subjects with pathologically proven DN, so our study population was unable to represent the entire spectrum of patients with DKD. The majority of our subjects showed significant decline in kidney function. We believe that rapidly progressive DKD is the area in need of investigation for novel therapeutic interventions. A major strength of our study is that this is the first study examining the expressions of biomarkers in human tissue with pathologically proven DN, and validating correlations between tubular expressions of NGAL and KIM-1, and the progression of DKD.
Please cite this article as: Hwang S, et al. Tissue expression of tubular injury markers is associated with renal function decline in diabetic nephropathy. Journal of Diabetes and Its Complications (2017), https://doi.org/10.1016/j.jdiacomp.2017.08.009
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S. Hwang et al. / Journal of Diabetes and Its Complications xxx (2017) xxx–xxx
Table 5 Correlation of tubular expressions with serum and urine levels of NGAL and KIM-1. Variables
Tubular expression of NGAL Tubular expression of KIM-1
a
uNGAL/Cr
b
a
sNGAL
uKIM-1/Cr
β
p
β
p
0.529
0.052
0.247
0.322
b
sKIM-1
β
p
β
p
0.878
b0.001
0.366
0.135
Abbreviations: uNGAL, urine neutrophil gelatinase-associated lipocalin; sNGAL, serum neutrophil gelatinase-associated lipocalin; uKIM-1, urine kidney injury molecule-1; sKIM-1, serum kidney injury molecule-1. a Urine samples were available in 18 subjects. b Serum samples were available in 14 subjects.
In conclusion, the present study demonstrated that tubular expressions of NGAL and KIM-1, tubular injury markers, were closely associated with GFR decline slopes in patients with pathologically proven DN. This finding suggests that tubular injury has a key role in the pathogenesis of DKD in patients at a high risk for kidney disease progression. Further studies are warranted to determine whether tubular injury can be a therapeutic target in DKD. Acknowledgements We thank Kyung Yi Choi, Ji Woo Kim, and Woo Hyun Lee of the Samsung Biomedical Research Institute for providing technical assistance. Grants This study was supported by a grant from the Samsung Biomedical Research Institute Grant (Grant No: OTC 1601971). References 1. Gregg EW, Li Y, Wang J, Burrows NR, Ali MK, Rolka D, et al. Changes in diabetes-related complications in the United States, 1990–2010. N Engl J Med. 2014;370:1514-23. 2. Pavkov ME, Knowler WC, Lemley KV, Mason CC, Myers BD, Nelson RG. Early renal function decline in type 2 diabetes. Clin J Am Soc Nephrol. 2012;7:78-84. 3. Berhane AM, Weil EJ, Knowler WC, Nelson RG, Hanson RL. Albuminuria and estimated glomerular filtration rate as predictors of diabetic end-stage renal disease and death. Clin J Am Soc Nephrol. 2011;6:2444-51. 4. Keane WF, Brenner BM, de Zeeuw D, Grunfeld JP, McGill J, Mitch WE, et al. The risk of developing end-stage renal disease in patients with type 2 diabetes and nephropathy: the RENAAL study. Kidney Int. 2003;63:1499-507. 5. Yokoyama H, Kanno S, Takahashi S, Yamada D, Itoh H, Saito K, et al. Determinants of decline in glomerular filtration rate in nonproteinuric subjects with or without diabetes and hypertension. Clin J Am Soc Nephrol. 2009;4:1432-40. 6. Lewis EJ, Hunsicker LG, Clarke WR, Berl T, Pohl MA, Lewis JB, et al. Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes. N Engl J Med. 2001;345:851-60. 7. Hou FF, Zhang X, Zhang GH, Xie D, Chen PY, Zhang WR, et al. Efficacy and safety of benazepril for advanced chronic renal insufficiency. N Engl J Med. 2006;354: 131-40. 8. Hovind P, Rossing P, Tarnow L, Smidt UM, Parving HH. Progression of diabetic nephropathy. Kidney Int. 2001;59:702-9. 9. Thakar CV, Christianson A, Himmelfarb J, Leonard AC. Acute kidney injury episodes and chronic kidney disease risk in diabetes mellitus. Clin J Am Soc Nephrol. 2011;6:2567-72. 10. Perkins BA, Ficociello LH, Ostrander BE, Silva KH, Weinberg J, Warram JH, et al. Microalbuminuria and the risk for early progressive renal function decline in type 1 diabetes. J Am Soc Nephrol. 2007;18:1353-61. 11. Niewczas MA, Gohda T, Skupien J, Smiles AM, Walker WH, Rosetti F, et al. Circulating TNF receptors 1 and 2 predict ESRD in type 2 diabetes. J Am Soc Nephrol. 2012;23:507-15. 12. Sun L, Kanwar YS. Relevance of TNF-alpha in the context of other inflammatory cytokines in the progression of diabetic nephropathy. Kidney Int. 2015;88:662-5.
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Please cite this article as: Hwang S, et al. Tissue expression of tubular injury markers is associated with renal function decline in diabetic nephropathy. Journal of Diabetes and Its Complications (2017), https://doi.org/10.1016/j.jdiacomp.2017.08.009