- Email: [email protected]
Type 2 diabetic patients with diabetic retinopathy and concomitant microalbuminuria showed typical diabetic glomerulosclerosis and progressive renal dysfunction
Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M
Type 2 diabetic patients with diabetic retinopathy and concomitant microalbuminuria showed typical diabetic glomerulosclerosis and progressive renal dysfunction Tatsumi Moriya a,⁎, Madoka Matsubara a, Eriko Kishihara a, Yuki Yoshida a, Motoshi Ouchi b a b
Heath Care Center, Kitasato University, Kanagawa, Japan Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Tochigi, Japan
a r t i c l e
i n f o
Article history: Received 28 January 2016 Received in revised form 1 April 2016 Accepted 11 April 2016 Available online xxxx Keywords: Diabetic nephropathy Diabetic retinopathy Microalbuminuria Diabetic glomerulosclerosis Renal function Histological heterogeneity
a b s t r a c t Aims: To determine whether or not diabetic retinopathy (DR) in type 2 diabetic patients can predict the renal functional decline. Methods: We examined 32 normo-microalbuminuric type 2 diabetic patients by renal biopsy (23 men, age 49 ± 10 yrs) divided into two groups according to the presence (n = 19) or absence (n = 13) of DR. Electron microscopic morphometry including mesangial fractional volume [Vv(Mes/glom)] were performed and light microscopic tissues were categorized as: C1, normal/near normal renal structure; C2, typical diabetic glomerulopathy; C3, atypical injury patterns. Patients were followed up for 7.1 ± 3.8 years, and glomerular filtration rate (GFR) and urinary albumin excretion (UAE) measurements were taken annually. Results: Vv(Mes/glom) was larger in DR + than that in DR–. Vv(Mes/glom) positively correlated with the UAE if patients had DR. The patients with DR had a significant higher rate of C2 pattern compared to those in DR–. Among patients with DR and C2, GFR in microalbuminuria (n = 7) decreased while GFR in normoalbuminuria (n = 5) did not change during the observation. Conclusions: Type 2 diabetic patients with DR and C2 showed progressive renal dysfunction after they had microalbuminuria. DR and albuminuria should be considered to determine renal function decline in type 2 diabetic patients. © 2016 Published by Elsevier Inc.
1. Introduction The detailed clinical courses of diabetic retinopathy (DR) and diabetic nephropathy (DN) and their relationship remain unknown; and whether DR and DN are parallel to progress is also controversial, especially in type 2 diabetes because only a few small studies have demonstrated the relationships between DR and DN (Kanauchi, Kawano, Uyama, Shiiki, & Dohi, 1998). In addition, the outcome to the renin–angiotensin system inhibitors was shown to be different between these two complications (Mauer et al., 2009). Regarding renal histological changes, one study showed that renal histological injury such as diabetic glomerulosclerosis including the glomerular basement membrane (GBM) thickening and/or mesangial expansion became more severe according to the DR severity in normoalbuminuric patients with type 1 diabetes (Klein et al., 2005).
Conflicts of interest: None. ⁎ Corresponding author at: Health Care Center, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252–0373, Japan. Tel.: +81 42 778 7601; fax: +81 42 778 7778. E-mail address: [email protected] (T. Moriya).
However, it remains unknown in patients with type 2 diabetes whether or not histological changes of diabetic glomerulosclerosis are more severe with or without DR or according to the DR severity. Fioretto et al. (1996) showed that renal histological changes were heterogeneous in patients with type 2 diabetes with microalbuminuria and that proliferative DR, not simple DR, was seen in only microalbuminuric type 2 diabetes with typical glomerular lesions. We have shown that patients with type 2 diabetes with typical diabetic glomerulosclerosis resulted in progressive albuminuria increase and glomerular filtration rate (GFR) loss (Moriya et al., 2014). However, that study (Moriya et al., 2014) did not show that DR itself was related to a renal histological injury pattern at the early stage of DN in patients with type 2 diabetes. In addition, it remains unknown whether or not renal functional decline occurs according to DR severity in patients with type 2 diabetes. Thirty-eight Caucasian patients with type 2 diabetes and macroalbuminuria showed a higher rate of GFR decline during 6 years of observation when the patients had DR compared with the patients without DR (Trevisan et al., 2002). In Chinese populations, the reduction of eGFR of more than 50% or the progression to eGFR b15 ml/min/1.73 m 2 or end-stage renal disease were predicted in
http://dx.doi.org/10.1016/j.jdiacomp.2016.04.007 1056-8727/© 2016 Published by Elsevier Inc.
Please cite this article as: Moriya, T., et al., Type 2 diabetic patients with diabetic retinopathy and concomitant microalbuminuria showed typical diabetic glomerulosclerosis and..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.04.007
2
T. Moriya et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
patients with type 2 diabetes having microalbuminuria or DR compared with the patients with no complications (Tong et al., 2007). More recently, the Japan Diabetic Complications Study (JDCS) (Moriya et al., 2013) showed that the presence of both DR and microalbuminuria predicted progressive GFR decline during an 8-year follow-up. However, those reports (Moriya et al., 2013; Tong et al., 2007) did not reveal any renal histological evidence in progressed patients. Therefore, to clarify the effects of DR on the course of the renal functional changes during an extensive, long-term follow-up, we examined 32 patients with type 2 diabetes with renal-biopsyproven DN according to the presence or absence of DR. 2. Subjects, materials, and methods 2.1. Type 2 diabetic patients Since 1998, normotensive patients with type 2 diabetes without macroalbuminuria, hematuria, or renal dysfunction and without any evidence suggesting atherosclerotic diseases were recruited at the outpatient clinic of Kitasato University Hospital. We excluded the patients whose biopsies were performed because of a clinical indication from the previous study. Patients who were also excluded from this initial recruitment were: patients who were receiving antihypertensive drugs; patients with a past history of any malignant, cerebrovascular, or cardiovascular diseases; and patients with recurrent infection. All the patients were fully informed about the trial, and 43 Japanese patients with type 2 diabetes, who provided written informed consent to participate in this study, as well as for the writing and publication of this report, received percutaneous renal biopsies at the Kitasato University Hospital. Of those 43 patients, 11 were excluded after the renal biopsy results were examined. One patient was excluded due to insufficient biopsy material for an accurate diagnosis, and the remaining 10 patients revealed evident IgA (immunoglobulin A) nephropathy with or without diabetic changes. The remaining 32 patients showed no evidence of non-diabetic renal glomerular or tubular/interstitial changes. Some cases were used for parts of previous studies (Moriya, Moriya, Yajima, Steffes, & Mauer, 2002; Moriya, Tanaka, Hosaka, Hirasawa, & Fujita, 2008; Moriya et al., 2012, 2014). There were 9 women and 23 men (49 ± 10 years) with a known diabetic duration of 13 ± 8 years. Normoalbuminuria was defined as a urinary albumin excretion (UAE) of less than 20 μg/min where as microalbuminuria was defined as a UAE of between 20 and 200 μg/min using 4-h collected urine samples at the GFR measurement described below. Clinical examinations were performed along with a renal biopsy and followed up annually after the renal biopsy. The protocol for this study was approved by the Research Ethics Committee of Kitasato University School of Medicine. 2.2. Laboratory and clinical measurements HbA1c was measured by high-performance liquid chromatography (HPLC). The value for HbA1c (%) was estimated as the National Glycohemoglobin Standardization Program (NGSP) equivalent value (%) calculated by the formula HbA1c (%) = HbA1c (the Japan Diabetes Society [JDS]) (%) + 0.4%, considering the relational expression of HbA1c (JDS) (%) measured by the previous Japanese standard substance and measurement methods and HbA1c (NGSP) (Committee of the Japan Society on the Diagnostic Criteria of Diabetes Mellitus et al., 2010). The GFR was measured by the plasma clearance of unlabeled iohexol (Jin, Moriya, Tanaka, Matsubara, & Fujita, 2006; Krutzen, Back, Nilsson-Ehle, & Nilsson-Ehle, 1984; Moriya et al., 2012). Briefly, 5 ml iohexol (3,235 mg) was injected intravenously, and blood samples were drawn 2, 3, and 4 h after the injection. The plasma concentration of iohexol was measured by HPLC, and the GFRs were calculated with the slope of the terminal plasma disappearance curve of iohexol in the Central Laboratory in the Kitasato University
Hospital. The average coefficients of variation (CV) was 10.3 ± 3.1% in repeated measurements of iohexol-based GFRs in 16 patients without showing significant changes in renal function. Although it is not certain to what extent the variation derives from physiological variation, this value gives rough estimates on the possible maximal fluctuation accompanied by determining iohexol-based GFR (Moriya et al., 2012). Urinary albumin was measured by turbidimetric immunoassay using 4-h urine collections at the GFR measurement. Blood pressure (BP) was measured during the initial hospitalization for the renal biopsy and at the outpatient visits. Hypertension was defined as systolic BP ≥ 130 mmHg, diastolic BP ≥ 85 mmHg and/or patients receiving treatment with antihypertensive drugs. During the follow-up period, patients developing hypertension were prescribed antihypertensives to achieve optimal BP control (under 130/80 mmHg). None required antihypertensives at the initial evaluation. However, 3 normoalbuminuric and 5 microalbuminuric patients were prescribed renin–angiotensin–aldosterone inhibitors (RAS inhibitors) during the observation period. The level of albuminuria in 8 cases did not change and microalbuminuria in 5 cases did not return to normoalbuminuria during the observation period, although RAS inhibitors were prescribed (data not shown). 2.3. Morphometric analysis of renal biopsies For light microscopic morphometric analysis, renal biopsy tissues from diabetic patients were fixed in 10% buffered formalin and stained with periodic acid-Schiff stain. The mean glomerular volume (MGV) was determined by the light microscopic examination of the sections at an approximate magnification of × 150 using the point-counting method of Weibel and Gometz (Weibel, 1979). At least 15 glomerular profiles (24 ± 10, mean ± SD) were measured for each patient. In addition, renal injury patterns of light microscopy tissues were categorized according to the previous study as follows (Fioretto et al., 1996). Category I (C1): normal or near normal renal structure. These patients had biopsies that were normal or showed very mild mesangial expansion, tubulointerstitial changes or arteriolar hyalinosis in any combination. Category II (C2): typical diabetic glomerulopathy with parallel changes in the tubulointerstitium and vessels. These patients had established diabetic lesions with balanced severity of glomerular, tubulointerstitial, and arteriolar changes. Category III (C3): atypical injury patterns. These patients had absent or mild diabetic glomerulopathy associated with disproportionately severe tubulointerstitial injury, and/or arteriolar hyalinosis, and/or global glomerular sclerosis, including: (a) tubular atrophy, tubular basement membrane thickening and reduplication, and interstitial fibrosis (tubulointerstitial lesions); (b) advanced glomerular arteriolar hyalinosis commonly associated with atherosclerosis of larger vessels; and (c) global glomerular sclerosis (N 25%) in the presence of absent or mild mesangial expansion. The categorization was performed by 4 or 5 observers (T. M., A. Tsuchiya, S. Wakakura, A. Hayashi, and S. Okizaki) who were blind to the identity of the patients. For electron microscopic morphometric analysis, kidney tissues were cut into cubes (approximately 1 mm 3), fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and postfixed in osmium tetroxide. These specimens were dehydrated in graded series of ethanol and embedded in Quetol 812 (Nissin EM, Inc., Tokyo, Japan). All specimens were cut into thick and 80–90 nm ultrathin sections and studied using a JEOL CX 100 transmission electron microscope (JEOL, Tokyo, Japan) in the Kitasato Bio-Imaging Center. Routine stereologic techniques, previously described (Chavers, Bilous, Ellis, Steffes, & Mauer, 1989; Fioretto, Steffes, & Mauer, 1994; Jensen, Gundersen, & Osterby, 1979; Mauer et al., 1984; Moriya et al., 2002),
Please cite this article as: Moriya, T., et al., Type 2 diabetic patients with diabetic retinopathy and concomitant microalbuminuria showed typical diabetic glomerulosclerosis and..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.04.007
T. Moriya et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
3
Table 1 Clinical characteristics, morphometric analysis, and histological injury patterns for the patients with and without diabetic retinopathy.
Number (M/F) Age (yrs) BMI (kg/m2) Known duration (yrs) HbA1c (%) (mmol/mol) (NGSP/IFCC) DR (N/S/P) (number) SBP (mmHg) DBP (mmHg) Iohexol-GFR (ml/min/1.73 m2) UAE (μg/min) Normoalbuminuria/Microalbuminuria MGV (×106 μm3) GBM width (nm) Vv(Mes/glom) Sv(PGBM/glom) (μm2/μm3) Total Mes/glom (×106 μm3) Filtration S/G (μm2) C1/C2/C3/unclassified
DR–
DR+
P-value
13 (10/3) 50 ± 10 22.7 ± 2.3 11 ± 7 7.9 ± 2.1/63.3 ± 22.5 13/0/0 115 ± 4 71 ± 12 123.7 ± 26.8 15.1 (0.0–180.2) 7/6 3.5 ± 0.8 681 ± 137 0.21 ± 0.04 0.12 ± 0.04 0.72 ± 0.18 0.45 ± 0.13 3/1/8/1
19 (13/6) 48 ± 10 23.2 ± 4.5 14 ± 8 7.7 ± 1.7/61.2 ± 19.0 0/14/5 123 ± 4 74 ± 10 122.8 ± 39.5 10.9 (0.59–82.4) 12/7 2.9 ± 0.8 737 ± 99 0.27 ± 0.02 0.10 ± 0.03 0.79 ± 0.31 0.30 ± 0.11 2/12/4/1
0.599 0.548 0.735 0.437 0.773/0.773 ND 0.162 0.507 0.945 0.144 0.599 0.086 0.260 0.011 0.122 0.559 0.004 0.018 (χ2 = 10.07)
Data are n or means ± SD. DR, diabetic retinopathy; M, male; F, female; BMI, body mass index; NGSP/IFCC, National Glycohemoglobin Standardization Program/International Federation of Clinical Chemistry; N/S/P, none/simple/preproliferative or proliferative; SBP, systolic blood pressure; DBP, diastolic blood pressure; GFR, glomerular filtration rate; MGV, mean glomerular volume; GBM, glomerular basement membrane; Vv(Mes/glom), volume fraction of mesangium; Sv(PGBM/glom), surface density of peripheral GBM; Total Mes/glom, total mesangium per glomerulus; S/G, surface/glomerulus; ND, not done for selection criterion.
Total Mes=glom ¼ MGV VvðMes=glomÞ Filtration S=G ¼ MGV SvðPGBM=glomÞ
2.4. Assessment of DR The presence and severity of DR were determined by qualified ophthalmologists by mydriatic indirect ophthalmoscopic examination and slit lamp biomicroscopic fundus examination using precorneal lens using the international DR and diabetic macular edema disease scales with minor modifications (Wilkinson et al., 2003). Severity of DR was categorized following the international clinical diabetic retinopathy severity scales into 5 categories as “no retinopathy” (equivalent to the Early Treatment of Diabetic Retinopathy Study [ETDRS] scale level 10), “mild non-proliferative DR” (stage 1; equivalent to ETDRS level 20), “moderate non-proliferative DR” (stage 2; equivalent to ETDRS levels 35, 43, and 47), “severe non-proliferative DR” (stage 3; equivalent to ETDRS levels 53A–53E) and “proliferative DR” (stage 4; equivalent to ETDRS levels 61 or above) (Wilkinson et al., 2003). In the present study, stages 1–2 were considered as simple DR, and stages 3 and 4 were categorized as preproliferative and proliferative DR, respectively. History of ocular surgery (e.g., cataract, glaucoma, and vitreoretinal surgery) was also taken into account.
test. All P values are two-sided with values less than 0.05 considered as indicating statistical significance. All statistical analyses and data management were conducted using JMP ver. 10 (SAS Institute Inc., Cary, NC). 3. Results 3.1. Demographic data and clinical characteristics between the two groups Thirty-two patients were divided into two groups according to the absence or presence of DR. Nineteen patients had DR (14, simple; 5, preproliferative or proliferative). Baseline clinical characteristics between these two diabetic groups, such as age, BMI (body mass index), known duration of diabetes, HbA1c, SBP (systolic blood pressure), DBP (diastolic blood pressure), GFR measured by iohexol injection and UAE did not exhibit any significant differences (Table 1). None required antihypertensives at the initial evaluation.
Vv(Mes/glom)
were used to measure the glomerular basement membrane (GBM) width, mesangial fractional volume [Vv(Mes/glom)], and the surface density of the peripheral GBM [Sv(PGBM/glom)]. Briefly, GBM width was measured using the orthogonal intercept method (Jensen et al., 1979), Vv(Mes/glom) by point counting (Chavers et al., 1989; Fioretto et al., 1994; Mauer et al., 1984; Moriya et al., 2002) and Sv(PGBM/glom) by using the line-intercept method (Chavers et al., 1989; Fioretto et al., 1994; Mauer et al., 1984; Moriya et al., 2002). We calculated total mesangium per glomerulus (Total Mes/glom) and filtration surface per glomerulus (Filtration S/G) using the following formulae.
UAE (µg/min) 2.5. Statistical analyses Data are presented as mean ± SD. Relationships between renal structure and function were analyzed using regression analysis. Group comparisons were performed using either the t-test or a non-parametric
Fig. 1. The relationship between the UAE and electron microscopic morphometry. The volume fraction of the mesangium [Vv(Mes/glom)] was significantly and positively correlated with the baseline UAE when the patients had diabetic retinopathy (DR) (closed circles) (r = 0.55, P b 0.05). The patients without DR (open circles) did not show any significant correlations between the UAE and any morphometric data.
Please cite this article as: Moriya, T., et al., Type 2 diabetic patients with diabetic retinopathy and concomitant microalbuminuria showed typical diabetic glomerulosclerosis and..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.04.007
4
T. Moriya et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
3.2. Morphometric analysis, renal histological injury patterns and renal structural–functional relationships MGV, GBM width, Sv(PGBM/glom), and total Mes/glom were not different between the two groups (Table 1). The values of Vv(Mes/glom) in DR+ were significantly larger than that in DR–. Filtration S/G in DR+ was significantly smaller than that in DR–. The histological injury patterns were significantly different between the two groups. Proportion of C2 in DR+ was significantly increased compared to DR–. We subsequently compared the renal structural–functional relationships according to the presence or absence of DR. Vv(Mes/glom) was significantly and positively correlated with the UAE at biopsy among the patients with DR (Fig. 1). In addition, the histological pattern was not related to the albuminuria category. 3.3. Follow-up data of renal function in typical histological injury pattern and stepwise regression analysis Previously, we indicated that typical diabetic glomerular injury pattern (C2) showed progressive urinary albumin increase and renal function decline (Moriya et al., 2014). In addition, patients with concomitant microalbuminuria and DR showed progressive renal function decline (Moriya et al., 2013). However, it is still unknown whether or not the C2 pattern, the presence of DR and microalbuminuria are related and which combinations affect renal function decline. Therefore, we analyzed the course of renal function in normoalbuminuric and microalbuminuric patients with the C2 pattern and DR (Table 2). Noteworthy, all of the normoalbuminuric patients and 7 of 8 microalbuminuric patients with the C2 pattern had DR among all the patients (Table 2). There were no significant differences in any of the morphometric data between the two groups (Table 2), and baseline GFR did not differ between the two groups. After 7.1 ± 3.8 years of follow-up, UAE did not change from baseline to follow-up in either group. However, GFR in microalbuminuria with C2 had significantly decreased compared to the baseline, while GFR did not change from baseline to follow-up in normoalbuminuria with the C2 pattern (Table 2). Additionally, baseline GFR and UAE in either group did not have any significant differences compared to normoalbuminuria or microalbuminuria with histological patterns out of C2 (data not shown). On the other hand, the patients with DR, but not with C2 pattern (n = 12) and patients without both C2 pattern and DR (n = 6), showed no significant changes of UAE and GFR between the baseline and the follow-up. Vv(Mes/glom) in these 2 groups (0.21 ± 0.03, 0.21 ± 0.05, respectively) and total Mes/glom (0.72 ± 0.19, 0.51 ± 0.11, respectively) were significantly smaller than both normoalbuminuric and microalbuminuric patients with C2 pattern. We subsequently tested what factors, such as mesangial expansion, DR, albuminuria or histological injury pattern mostly
Table 3 Stepwise regression analysis. Independent variables
F-value
P-value
(Dependent variable: Final GFR) Vv(Mes/glom) DR only Microalbuminuria only Histological injury pattern only DR and microalbuminuria
3.360 0.708 2.766 1.934 7.240
0.08 0.41 0.11 0.18 0.01
GFR, glomerular filtration rate; Vv(Mes/glom), volume fraction of mesangium; DR, diabetic retinopathy.
affected the follow-up GFR. The categorization using both DR and microalbuminuria mostly affected GFR at the final observation (Table 3). The categorization of the presence of only DR, or only microalbuminuria, or mesangial expansion, did not significantly affect the final GFR. 4. Discussion We have shown in patients with type 2 diabetes and DR that typical diabetic glomerulosclerosis was significantly present, and GFR decline occurred when they had concomitant microalbuminuria. The study procedures allowed the analyses of structural–functional relationships and renal functional outcomes. It was already shown that renal function loss strikingly occurred in patients with type 2 diabetes with both DR and microalbuminuria using a large number of Japanese patients (Moriya et al., 2013). In Chinese populations, the renal outcome was obviously increased when both DR and microalbuminuria or macroalbuminuria were present (Tong et al., 2007). However, these studies did not show any renal histological evidence in the patients with renal function loss (Moriya et al., 2013; Tong et al., 2007). In terms of relationships between DR and renal histology, the presence of DR has revealed higher incidence of Kimmelstiel–Wilson nodules in patients with type 2 diabetes with hypertension and macroalbuminuria (Schwartz, Lewis, Leonard-Martin, Lewis, & Batlle, 1998). On the other hand, the mesangial volume fraction correlated with DR severity when the patients had hypertension from normoalbuminuria to macroalbuminuria in type 1 diabetes (Chavers, Mauer, Ramsay, & Steffes, 1994). Moreover, renal histological changes were more severe in Caucasian normoalbuminuric patients with type 1 diabetes with DR than those without DR (Klein et al., 2005). However, it remains unknown whether or not DR is related to renal histological changes at the early stage of DN, especially in type 2 diabetes; although one study, utilizing microalbuminuric type 2 diabetes, showed that only patients with typical diabetic glomerulosclerosis had proliferative DR (Fioretto et al., 1996).
Table 2 Follow-up data of renal function between normoalbuminuria and microalbuminuria with typical histological injury patterns and DR.
Number DR UAE at baseline (μg/min) UAE at follow-up (μg/min) P-value (UAE at baseline vs. follow-up) Iohexol-GFR at baseline (ml/min/1.73 m2) Iohexol-GFR at follow-up (ml/min/1.73 m2) P-value (GFR at baseline vs. follow-up) MGV (×106 μm3) GBM width (nm) Vv(Mes/glom) Sv(PGBM/glom) (μm2/μm3) Total Mes/glom (×106 μm3) Filtration S/G (μm2)
Normoalbuminuria with C2
Microalbuminuria with C2
P-value
5 5 5.4 (5.1–14.6) 15.4 (4.6–244.0) 0.834 107.3 ± 19.8 120.6 ± 23.4 0.821 3.2 ± 1.3 810 ± 118 0.31 ± 0.07 0.09 ± 0.03 1.00 ± 0.41 0.26 ± 0.10
7 7 48.6 (28.3–52.0) 71.9 (24.4–428.3) 0.848 130.6 ± 54.7 80.9 ± 42.0 0.048 3.1 ± 0.3 743 ± 117 0.29 ± 0.06 0.09 ± 0.04 0.92 ± 0.21 0.30 ± 0.16
ND ND ND ND 0.925 0.309 0.851 0.470 0.653 0.958 0.695 0.666
Data are n, median (interquartile range) or means ± SD. UAE, urinary albumin excretion; GFR, glomerular filtration rate; ND, not done for selection criterion.
Please cite this article as: Moriya, T., et al., Type 2 diabetic patients with diabetic retinopathy and concomitant microalbuminuria showed typical diabetic glomerulosclerosis and..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.04.007
T. Moriya et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
In the present study, we described the histological injury patterns from normoalbuminuria to microalbuminuria in type 2 diabetes. All microalbuminuric patients with DR, and 5 of 7 normoalbuminuria patients with DR, had the C2 pattern. Furthermore, the patients with DR had significant increases in the mesangial volume fraction as revealed by electron microscopic morphometry compared with that in patients without DR. These data suggested that typical diabetic glomerulosclerosis and renal histological severity related to DR in patients with type 2 diabetes and was compatible with that seen in patients with type 1 diabetes with DR (Klein et al., 2005). Our previous study did not show any correlation between urinary albumin and histological changes including Vv(Mes/glom) at the early stage of DN (Moriya et al., 2002). That study purported that renal structural–functional relationships were thought not to be present at the early stage of DN in patients with type 2 diabetes. However, in the present study, we discovered that there was a positive correlation between the UAE and Vv(Mes/glom) in the patients with DR. This phenomenon suggested that renal structural–functional relationships became evident even at the early stage of DN if the patients had DR. This phenomenon might depend on the fact that renal histological changes, such as diabetic glomerulosclerosis, were more severe in patients with DR, as evidenced in the present study and in another previous study (Klein et al., 2005). More recently, a link between the quantitative assessment of the retinal vessel caliber and the change in the glomerulopathy index, including mesangial expansion and GBM thickening during 5 years of follow-up, was reported in normotensive normoalbuminuric Caucasian type 1 diabetic patients (Klein et al., 2010). Change in the retinal vessel diameter has been speculated to reflect inflammation, endothelial dysfunction, and prevalence and incidence of DR (Nguyen & Wong, 2009). Therefore, DR might reflect renal histological severity as diabetic glomerulosclerosis regardless of albuminuria. We can interpret that the patients without DR make the renal structural– functional relationships complicated in patients with type 2 diabetes. We need to examine other renal histological changes than glomerular changes including tubulointerstitial and vascular damage in these types of cases. In renal function, there were no significant differences of GFR measured by iohexol injection between normoalbuminuria with C2 and microalbuminuria with C2 at the baseline. GFR in microalbuminuria with C2 significantly decreased at the follow-up compared to the baseline. This phenomenon might show that typical diabetic glomerulosclerosis resulted in a GFR decline over several years of follow-up through microalbuminuria, which is compatible with our previous study (Moriya et al., 2014). On the other hand, we have shown that histological severity, not related to initial UAE, predicted an increase in UAE 5–6 years later (Moriya et al., 2008). However, Vv(Mes/glom) itself did not affect the GFR at the follow-up in the present study, while in microalbuminuric Caucasian type 1 diabetic patients, the rate of annual GFR decline was related to the renal histological change (Rudberg & Osterby, 1997). Moreover, the classifications using only microalbuminuria or only DR did not affect the final GFR. Therefore, the present analysis might indicate the consideration that a change in both albuminuria and DR predicts a GFR decline, which is compatible with the previous JDCS (Moriya et al., 2013) and suggests the necessity to refer all such patients to ophthalmologists, especially when they are in the microalbuminuric stage. The limitation of this study was that the relatively small population of patients. Research-related renal biopsies can almost never be performed in our country at the early stage of DN. However, it was evidenced that DR was related to typical histological injury patterns and predict renal function loss when patients had microalbuminuria, as shown in the JDCS (Moriya et al., 2013) using renal biopsy together with the more accurate iohexol-GFR method. Further follow-up study is warranted to examine a larger population of
5
patients over a longer period of time. Another limitation was that there might be selection bias of the patients because there were no hypertensive patients at the baseline. Hypertension is known to have effects on retinopathy since long-term elevation of BP has been shown to be associated to retinopathy (Avery et al., 2012). It has also been reported that the severity of DR and nephropathy were related in the presence of hypertension (Chavers et al., 1994). However, the present study focused on the early stage of diabetic nephropathy. In addition, we wanted to avoid the effect of BP on the renal function course because we planned to test the effect of DR itself on renal function. Further study is warranted to examine the effects of BP on DR in Japanese type 2 diabetic patients. In summary, in the normo- and microalbuminuric patients with type 2 diabetes with a definite histological diagnosis of DN, renal structural–functional relationships became more obvious in the presence of DR, and microalbuminuria with DR showed typical diabetic glomerulosclerosis and development of GFR decline. Therefore, both DR and microalbuminuria should be considered to determine whether or not the renal function progresses in patients with type 2 diabetes.
Acknowledgements The authors thank Eriko Oda and Noriko Nemoto of the Kitasato Bio-Imaging Center for their technical assistance. The study was presented in part at the 71st Annual Scientific Meeting of the American Diabetes Association, 2011. We also thank Robert E. Brandt, Founder, CEO, and CME, of MedEd Japan, for editing and formatting the manuscript.
References Avery, C. L., Kucharska-Newton, A., Monda, K. L., Richy Sharrett, A., Mosley, T. H., Klein, B. E., ... Klein, R. (2012). Impact of long-term measures of glucose and blood pressure on the retinal microvasculature. Atherosclerosis, 225, 412–417. Chavers, B. M., Bilous, R. W., Ellis, E. N., Steffes, M. W., & Mauer, S. M. (1989). Glomerular lesions and urinary albumin excretion in type I diabetes without overt proteinuria. The New England Journal of Medicine, 320, 966–970. Chavers, B. M., Mauer, S. M., Ramsay, R. C., & Steffes, M. W. (1994). Relationship between retinal and glomerular lesions in IDDM patients. Diabetes, 43, 441–446. Committee of the Japan Society on the Diagnostic Criteria of Diabetes Mellitus, Seino, Y., Nanjo, K., Tajima, N., Kadowaki, T., Kashiwagi, A., Araki, E., et al. (2010). Report of the committee on the classification and diagnostic criteria of diabetes mellitus. Journal of Diabetes Investigation, 1, 212–228. Fioretto, P., Mauer, M., Brocco, E., Velussi, M., Frigato, F., Muollo, B., ... Nosadini, R. (1996). Patterns of renal injury in NIDDM patients with microalbuminuria. Diabetologia, 39, 1569–1576. Fioretto, P., Steffes, M. W., & Mauer, M. (1994). Glomerular structure in nonproteinuric IDDM patients with various levels of albuminuria. Diabetes, 43, 1358–1364. Jensen, E. B., Gundersen, H. J., & Osterby, R. (1979). Determination of membrane thickness distribution from orthogonal intercepts. Journal of Microscopy, 115, 19–33. Jin, Y., Moriya, T., Tanaka, K., Matsubara, M., & Fujita, Y. (2006). Glomerular hyperfiltration in non-proteinuric and non-hypertensive Japanese type 2 diabetic patients. Diabetes Research and Clinical Practice, 71, 264–271. Kanauchi, M., Kawano, T., Uyama, H., Shiiki, H., & Dohi, K. (1998). Discordance between retinopathy and nephropathy in type 2 diabetes. Nephron, 80, 171–174. Klein, R., Knudtson, M. D., Klein, B. E., Zinman, B., Gardiner, R., Suissa, S., ... Mauer, M. (2010). The relationship of retinal vessel diameter to changes in diabetic nephropathy structural variables in patients with type 1 diabetes. Diabetologia, 53, 1638–1646. Klein, R., Zinman, B., Gardiner, R., Suissa, S., Donnelly, S. M., Sinaiko, A. R., ... Mauer, M. Renin-Angiotensin System Study. (2005). The relationship of diabetic retinopathy to preclinical diabetic glomerulopathy lesions in type 1 diabetic patients: The Renin-Angiotensin System Study. Diabetes, 54, 527–533. Krutzen, E., Back, S. E., Nilsson-Ehle, I., & Nilsson-Ehle, P. (1984). Plasma clearance of a new contrast agent, iohexol: A method for the assessment of glomerular filtration rate. The Journal of Laboratory and Clinical Medicine, 104, 955–961. Mauer, S. M., Steffes, M. W., Ellis, E. N., Sutherland, D. E., Brown, D. M., & Goetz, F. C. (1984). Structural-functional relationships in diabetic nephropathy. The Journal of Clinical Investigation, 74, 1143–1155. Mauer, M., Zinman, B., Gardiner, R., Suissa, S., Sinaiko, A., Strand, T., ... Klein, R. (2009). Renal and retinal effects of enalapril and losartan in type 1 diabetes. The New England Journal of Medicine, 361, 40–51.
Please cite this article as: Moriya, T., et al., Type 2 diabetic patients with diabetic retinopathy and concomitant microalbuminuria showed typical diabetic glomerulosclerosis and..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.04.007
6
T. Moriya et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
Moriya, T., Moriya, R., Yajima, Y., Steffes, M. W., & Mauer, M. (2002). Urinary albumin as an indicator of diabetic nephropathy lesions in Japanese type 2 diabetic patients. Nephron, 91, 292–299. Moriya, T., Suzuki, Y., Inomata, S., Iwano, M., Kanauchi, M., & Haneda, M. (2014). Renal histological heterogeneity and functional progress in normoalbuminuric and microalbuminuric Japanese patients with type 2 diabetes. BMJ Open Diabetes Research & Care, 2, e000029. Moriya, T., Tanaka, K., Hosaka, T., Hirasawa, Y., & Fujita, Y. (2008). Renal structure as an indicator for development of albuminuria in normo- and microalbuminuric type 2 diabetic patients. Diabetes Research and Clinical Practice, 82, 298–304. Moriya, T., Tanaka, S., Kawasaki, R., Ohashi, Y., Akanuma, Y., Yamada, N., ... Katayama, S. (2013). Japan Diabetes Complications Study Group. Diabetic retinopathy and microalbuminuria can predict macroalbuminuria and renal function decline in Japanese type 2 diabetic patients: Japan Diabetes Complications Study. Diabetes Care, 36, 2803–2809. Moriya, T., Tsuchiya, A., Okizaki, S., Hayashi, A., Tanaka, K., & Shichiri, M. (2012). Glomerular hyperfiltration and increased glomerular filtration surface are associated with renal function decline in normo- and microalbuminuric type 2 diabetes. Kidney International, 81, 486–493. Nguyen, T. T., & Wong, T. Y. (2009). Retinal vascular changes and diabetic retinopathy. Current Diabetes Reports, 9, 277–283.
Rudberg, S., & Osterby, R. (1997). Decreasing glomerular filtration rate–An indicator of more advanced diabetic glomerulopathy in the early course of microalbuminuria in IDDM adolescents? Nephrology, Dialysis, Transplantation, 12, 1149–1154. Schwartz, M. M., Lewis, E. J., Leonard-Martin, T., Lewis, J. B., & Batlle, D. (1998). Renal pathology patterns in type II diabetes mellitus: Relationship with retinopathy. The Collaborative Study Group. Nephrology, Dialysis, Transplantation, 13, 2547–2552. Tong, P. C., Kong, A. P., So, W. Y., Yang, X., Ng, M. C., Ho, C. S., ... Cockram, C. S. (2007). Interactive effect of retinopathy and macroalbuminuria on all-cause mortality, cardiovascular and renal end points in Chinese patients with type 2 diabetes mellitus. Diabetic Medicine, 24, 741–746. Trevisan, R., Vedovato, M., Mazzon, C., Coracina, A., Iori, E., Tiengo, A., ... Del Prato, S. (2002). Concomitance of diabetic retinopathy and proteinuria accelerates the rate of decline of kidney function in type 2 diabetic patients. Diabetes Care, 25, 2026–2031. Weibel, E. R. (1979). Stereological methods. In E. R. Weibel (Ed.), Practical methods for biological morphometry, Vol 1. (pp. 9–62). London: Academic Press. Wilkinson, C. P., Ferris, F. L., III, Klein, R. E., Lee, P. P., Agardh, C. D., Davis, M., ... Verdaguer, J. T.Global Diabetic Retinopathy Project Group. (2003). Proposed international clinical diabetic retinopathy and diabetic macular edema disease severity scales. Ophthalmology, 110, 1677–1682.
Please cite this article as: Moriya, T., et al., Type 2 diabetic patients with diabetic retinopathy and concomitant microalbuminuria showed typical diabetic glomerulosclerosis and..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.04.007
Contents lists available at ScienceDirect
Journal of Diabetes and Its Complications j o u r n a l h o m e p a g e : W W W. J D C J O U R N A L . C O M
Type 2 diabetic patients with diabetic retinopathy and concomitant microalbuminuria showed typical diabetic glomerulosclerosis and progressive renal dysfunction Tatsumi Moriya a,⁎, Madoka Matsubara a, Eriko Kishihara a, Yuki Yoshida a, Motoshi Ouchi b a b
Heath Care Center, Kitasato University, Kanagawa, Japan Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Tochigi, Japan
a r t i c l e
i n f o
Article history: Received 28 January 2016 Received in revised form 1 April 2016 Accepted 11 April 2016 Available online xxxx Keywords: Diabetic nephropathy Diabetic retinopathy Microalbuminuria Diabetic glomerulosclerosis Renal function Histological heterogeneity
a b s t r a c t Aims: To determine whether or not diabetic retinopathy (DR) in type 2 diabetic patients can predict the renal functional decline. Methods: We examined 32 normo-microalbuminuric type 2 diabetic patients by renal biopsy (23 men, age 49 ± 10 yrs) divided into two groups according to the presence (n = 19) or absence (n = 13) of DR. Electron microscopic morphometry including mesangial fractional volume [Vv(Mes/glom)] were performed and light microscopic tissues were categorized as: C1, normal/near normal renal structure; C2, typical diabetic glomerulopathy; C3, atypical injury patterns. Patients were followed up for 7.1 ± 3.8 years, and glomerular filtration rate (GFR) and urinary albumin excretion (UAE) measurements were taken annually. Results: Vv(Mes/glom) was larger in DR + than that in DR–. Vv(Mes/glom) positively correlated with the UAE if patients had DR. The patients with DR had a significant higher rate of C2 pattern compared to those in DR–. Among patients with DR and C2, GFR in microalbuminuria (n = 7) decreased while GFR in normoalbuminuria (n = 5) did not change during the observation. Conclusions: Type 2 diabetic patients with DR and C2 showed progressive renal dysfunction after they had microalbuminuria. DR and albuminuria should be considered to determine renal function decline in type 2 diabetic patients. © 2016 Published by Elsevier Inc.
1. Introduction The detailed clinical courses of diabetic retinopathy (DR) and diabetic nephropathy (DN) and their relationship remain unknown; and whether DR and DN are parallel to progress is also controversial, especially in type 2 diabetes because only a few small studies have demonstrated the relationships between DR and DN (Kanauchi, Kawano, Uyama, Shiiki, & Dohi, 1998). In addition, the outcome to the renin–angiotensin system inhibitors was shown to be different between these two complications (Mauer et al., 2009). Regarding renal histological changes, one study showed that renal histological injury such as diabetic glomerulosclerosis including the glomerular basement membrane (GBM) thickening and/or mesangial expansion became more severe according to the DR severity in normoalbuminuric patients with type 1 diabetes (Klein et al., 2005).
Conflicts of interest: None. ⁎ Corresponding author at: Health Care Center, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252–0373, Japan. Tel.: +81 42 778 7601; fax: +81 42 778 7778. E-mail address: [email protected] (T. Moriya).
However, it remains unknown in patients with type 2 diabetes whether or not histological changes of diabetic glomerulosclerosis are more severe with or without DR or according to the DR severity. Fioretto et al. (1996) showed that renal histological changes were heterogeneous in patients with type 2 diabetes with microalbuminuria and that proliferative DR, not simple DR, was seen in only microalbuminuric type 2 diabetes with typical glomerular lesions. We have shown that patients with type 2 diabetes with typical diabetic glomerulosclerosis resulted in progressive albuminuria increase and glomerular filtration rate (GFR) loss (Moriya et al., 2014). However, that study (Moriya et al., 2014) did not show that DR itself was related to a renal histological injury pattern at the early stage of DN in patients with type 2 diabetes. In addition, it remains unknown whether or not renal functional decline occurs according to DR severity in patients with type 2 diabetes. Thirty-eight Caucasian patients with type 2 diabetes and macroalbuminuria showed a higher rate of GFR decline during 6 years of observation when the patients had DR compared with the patients without DR (Trevisan et al., 2002). In Chinese populations, the reduction of eGFR of more than 50% or the progression to eGFR b15 ml/min/1.73 m 2 or end-stage renal disease were predicted in
http://dx.doi.org/10.1016/j.jdiacomp.2016.04.007 1056-8727/© 2016 Published by Elsevier Inc.
Please cite this article as: Moriya, T., et al., Type 2 diabetic patients with diabetic retinopathy and concomitant microalbuminuria showed typical diabetic glomerulosclerosis and..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.04.007
2
T. Moriya et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
patients with type 2 diabetes having microalbuminuria or DR compared with the patients with no complications (Tong et al., 2007). More recently, the Japan Diabetic Complications Study (JDCS) (Moriya et al., 2013) showed that the presence of both DR and microalbuminuria predicted progressive GFR decline during an 8-year follow-up. However, those reports (Moriya et al., 2013; Tong et al., 2007) did not reveal any renal histological evidence in progressed patients. Therefore, to clarify the effects of DR on the course of the renal functional changes during an extensive, long-term follow-up, we examined 32 patients with type 2 diabetes with renal-biopsyproven DN according to the presence or absence of DR. 2. Subjects, materials, and methods 2.1. Type 2 diabetic patients Since 1998, normotensive patients with type 2 diabetes without macroalbuminuria, hematuria, or renal dysfunction and without any evidence suggesting atherosclerotic diseases were recruited at the outpatient clinic of Kitasato University Hospital. We excluded the patients whose biopsies were performed because of a clinical indication from the previous study. Patients who were also excluded from this initial recruitment were: patients who were receiving antihypertensive drugs; patients with a past history of any malignant, cerebrovascular, or cardiovascular diseases; and patients with recurrent infection. All the patients were fully informed about the trial, and 43 Japanese patients with type 2 diabetes, who provided written informed consent to participate in this study, as well as for the writing and publication of this report, received percutaneous renal biopsies at the Kitasato University Hospital. Of those 43 patients, 11 were excluded after the renal biopsy results were examined. One patient was excluded due to insufficient biopsy material for an accurate diagnosis, and the remaining 10 patients revealed evident IgA (immunoglobulin A) nephropathy with or without diabetic changes. The remaining 32 patients showed no evidence of non-diabetic renal glomerular or tubular/interstitial changes. Some cases were used for parts of previous studies (Moriya, Moriya, Yajima, Steffes, & Mauer, 2002; Moriya, Tanaka, Hosaka, Hirasawa, & Fujita, 2008; Moriya et al., 2012, 2014). There were 9 women and 23 men (49 ± 10 years) with a known diabetic duration of 13 ± 8 years. Normoalbuminuria was defined as a urinary albumin excretion (UAE) of less than 20 μg/min where as microalbuminuria was defined as a UAE of between 20 and 200 μg/min using 4-h collected urine samples at the GFR measurement described below. Clinical examinations were performed along with a renal biopsy and followed up annually after the renal biopsy. The protocol for this study was approved by the Research Ethics Committee of Kitasato University School of Medicine. 2.2. Laboratory and clinical measurements HbA1c was measured by high-performance liquid chromatography (HPLC). The value for HbA1c (%) was estimated as the National Glycohemoglobin Standardization Program (NGSP) equivalent value (%) calculated by the formula HbA1c (%) = HbA1c (the Japan Diabetes Society [JDS]) (%) + 0.4%, considering the relational expression of HbA1c (JDS) (%) measured by the previous Japanese standard substance and measurement methods and HbA1c (NGSP) (Committee of the Japan Society on the Diagnostic Criteria of Diabetes Mellitus et al., 2010). The GFR was measured by the plasma clearance of unlabeled iohexol (Jin, Moriya, Tanaka, Matsubara, & Fujita, 2006; Krutzen, Back, Nilsson-Ehle, & Nilsson-Ehle, 1984; Moriya et al., 2012). Briefly, 5 ml iohexol (3,235 mg) was injected intravenously, and blood samples were drawn 2, 3, and 4 h after the injection. The plasma concentration of iohexol was measured by HPLC, and the GFRs were calculated with the slope of the terminal plasma disappearance curve of iohexol in the Central Laboratory in the Kitasato University
Hospital. The average coefficients of variation (CV) was 10.3 ± 3.1% in repeated measurements of iohexol-based GFRs in 16 patients without showing significant changes in renal function. Although it is not certain to what extent the variation derives from physiological variation, this value gives rough estimates on the possible maximal fluctuation accompanied by determining iohexol-based GFR (Moriya et al., 2012). Urinary albumin was measured by turbidimetric immunoassay using 4-h urine collections at the GFR measurement. Blood pressure (BP) was measured during the initial hospitalization for the renal biopsy and at the outpatient visits. Hypertension was defined as systolic BP ≥ 130 mmHg, diastolic BP ≥ 85 mmHg and/or patients receiving treatment with antihypertensive drugs. During the follow-up period, patients developing hypertension were prescribed antihypertensives to achieve optimal BP control (under 130/80 mmHg). None required antihypertensives at the initial evaluation. However, 3 normoalbuminuric and 5 microalbuminuric patients were prescribed renin–angiotensin–aldosterone inhibitors (RAS inhibitors) during the observation period. The level of albuminuria in 8 cases did not change and microalbuminuria in 5 cases did not return to normoalbuminuria during the observation period, although RAS inhibitors were prescribed (data not shown). 2.3. Morphometric analysis of renal biopsies For light microscopic morphometric analysis, renal biopsy tissues from diabetic patients were fixed in 10% buffered formalin and stained with periodic acid-Schiff stain. The mean glomerular volume (MGV) was determined by the light microscopic examination of the sections at an approximate magnification of × 150 using the point-counting method of Weibel and Gometz (Weibel, 1979). At least 15 glomerular profiles (24 ± 10, mean ± SD) were measured for each patient. In addition, renal injury patterns of light microscopy tissues were categorized according to the previous study as follows (Fioretto et al., 1996). Category I (C1): normal or near normal renal structure. These patients had biopsies that were normal or showed very mild mesangial expansion, tubulointerstitial changes or arteriolar hyalinosis in any combination. Category II (C2): typical diabetic glomerulopathy with parallel changes in the tubulointerstitium and vessels. These patients had established diabetic lesions with balanced severity of glomerular, tubulointerstitial, and arteriolar changes. Category III (C3): atypical injury patterns. These patients had absent or mild diabetic glomerulopathy associated with disproportionately severe tubulointerstitial injury, and/or arteriolar hyalinosis, and/or global glomerular sclerosis, including: (a) tubular atrophy, tubular basement membrane thickening and reduplication, and interstitial fibrosis (tubulointerstitial lesions); (b) advanced glomerular arteriolar hyalinosis commonly associated with atherosclerosis of larger vessels; and (c) global glomerular sclerosis (N 25%) in the presence of absent or mild mesangial expansion. The categorization was performed by 4 or 5 observers (T. M., A. Tsuchiya, S. Wakakura, A. Hayashi, and S. Okizaki) who were blind to the identity of the patients. For electron microscopic morphometric analysis, kidney tissues were cut into cubes (approximately 1 mm 3), fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and postfixed in osmium tetroxide. These specimens were dehydrated in graded series of ethanol and embedded in Quetol 812 (Nissin EM, Inc., Tokyo, Japan). All specimens were cut into thick and 80–90 nm ultrathin sections and studied using a JEOL CX 100 transmission electron microscope (JEOL, Tokyo, Japan) in the Kitasato Bio-Imaging Center. Routine stereologic techniques, previously described (Chavers, Bilous, Ellis, Steffes, & Mauer, 1989; Fioretto, Steffes, & Mauer, 1994; Jensen, Gundersen, & Osterby, 1979; Mauer et al., 1984; Moriya et al., 2002),
Please cite this article as: Moriya, T., et al., Type 2 diabetic patients with diabetic retinopathy and concomitant microalbuminuria showed typical diabetic glomerulosclerosis and..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.04.007
T. Moriya et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
3
Table 1 Clinical characteristics, morphometric analysis, and histological injury patterns for the patients with and without diabetic retinopathy.
Number (M/F) Age (yrs) BMI (kg/m2) Known duration (yrs) HbA1c (%) (mmol/mol) (NGSP/IFCC) DR (N/S/P) (number) SBP (mmHg) DBP (mmHg) Iohexol-GFR (ml/min/1.73 m2) UAE (μg/min) Normoalbuminuria/Microalbuminuria MGV (×106 μm3) GBM width (nm) Vv(Mes/glom) Sv(PGBM/glom) (μm2/μm3) Total Mes/glom (×106 μm3) Filtration S/G (μm2) C1/C2/C3/unclassified
DR–
DR+
P-value
13 (10/3) 50 ± 10 22.7 ± 2.3 11 ± 7 7.9 ± 2.1/63.3 ± 22.5 13/0/0 115 ± 4 71 ± 12 123.7 ± 26.8 15.1 (0.0–180.2) 7/6 3.5 ± 0.8 681 ± 137 0.21 ± 0.04 0.12 ± 0.04 0.72 ± 0.18 0.45 ± 0.13 3/1/8/1
19 (13/6) 48 ± 10 23.2 ± 4.5 14 ± 8 7.7 ± 1.7/61.2 ± 19.0 0/14/5 123 ± 4 74 ± 10 122.8 ± 39.5 10.9 (0.59–82.4) 12/7 2.9 ± 0.8 737 ± 99 0.27 ± 0.02 0.10 ± 0.03 0.79 ± 0.31 0.30 ± 0.11 2/12/4/1
0.599 0.548 0.735 0.437 0.773/0.773 ND 0.162 0.507 0.945 0.144 0.599 0.086 0.260 0.011 0.122 0.559 0.004 0.018 (χ2 = 10.07)
Data are n or means ± SD. DR, diabetic retinopathy; M, male; F, female; BMI, body mass index; NGSP/IFCC, National Glycohemoglobin Standardization Program/International Federation of Clinical Chemistry; N/S/P, none/simple/preproliferative or proliferative; SBP, systolic blood pressure; DBP, diastolic blood pressure; GFR, glomerular filtration rate; MGV, mean glomerular volume; GBM, glomerular basement membrane; Vv(Mes/glom), volume fraction of mesangium; Sv(PGBM/glom), surface density of peripheral GBM; Total Mes/glom, total mesangium per glomerulus; S/G, surface/glomerulus; ND, not done for selection criterion.
Total Mes=glom ¼ MGV VvðMes=glomÞ Filtration S=G ¼ MGV SvðPGBM=glomÞ
2.4. Assessment of DR The presence and severity of DR were determined by qualified ophthalmologists by mydriatic indirect ophthalmoscopic examination and slit lamp biomicroscopic fundus examination using precorneal lens using the international DR and diabetic macular edema disease scales with minor modifications (Wilkinson et al., 2003). Severity of DR was categorized following the international clinical diabetic retinopathy severity scales into 5 categories as “no retinopathy” (equivalent to the Early Treatment of Diabetic Retinopathy Study [ETDRS] scale level 10), “mild non-proliferative DR” (stage 1; equivalent to ETDRS level 20), “moderate non-proliferative DR” (stage 2; equivalent to ETDRS levels 35, 43, and 47), “severe non-proliferative DR” (stage 3; equivalent to ETDRS levels 53A–53E) and “proliferative DR” (stage 4; equivalent to ETDRS levels 61 or above) (Wilkinson et al., 2003). In the present study, stages 1–2 were considered as simple DR, and stages 3 and 4 were categorized as preproliferative and proliferative DR, respectively. History of ocular surgery (e.g., cataract, glaucoma, and vitreoretinal surgery) was also taken into account.
test. All P values are two-sided with values less than 0.05 considered as indicating statistical significance. All statistical analyses and data management were conducted using JMP ver. 10 (SAS Institute Inc., Cary, NC). 3. Results 3.1. Demographic data and clinical characteristics between the two groups Thirty-two patients were divided into two groups according to the absence or presence of DR. Nineteen patients had DR (14, simple; 5, preproliferative or proliferative). Baseline clinical characteristics between these two diabetic groups, such as age, BMI (body mass index), known duration of diabetes, HbA1c, SBP (systolic blood pressure), DBP (diastolic blood pressure), GFR measured by iohexol injection and UAE did not exhibit any significant differences (Table 1). None required antihypertensives at the initial evaluation.
Vv(Mes/glom)
were used to measure the glomerular basement membrane (GBM) width, mesangial fractional volume [Vv(Mes/glom)], and the surface density of the peripheral GBM [Sv(PGBM/glom)]. Briefly, GBM width was measured using the orthogonal intercept method (Jensen et al., 1979), Vv(Mes/glom) by point counting (Chavers et al., 1989; Fioretto et al., 1994; Mauer et al., 1984; Moriya et al., 2002) and Sv(PGBM/glom) by using the line-intercept method (Chavers et al., 1989; Fioretto et al., 1994; Mauer et al., 1984; Moriya et al., 2002). We calculated total mesangium per glomerulus (Total Mes/glom) and filtration surface per glomerulus (Filtration S/G) using the following formulae.
UAE (µg/min) 2.5. Statistical analyses Data are presented as mean ± SD. Relationships between renal structure and function were analyzed using regression analysis. Group comparisons were performed using either the t-test or a non-parametric
Fig. 1. The relationship between the UAE and electron microscopic morphometry. The volume fraction of the mesangium [Vv(Mes/glom)] was significantly and positively correlated with the baseline UAE when the patients had diabetic retinopathy (DR) (closed circles) (r = 0.55, P b 0.05). The patients without DR (open circles) did not show any significant correlations between the UAE and any morphometric data.
Please cite this article as: Moriya, T., et al., Type 2 diabetic patients with diabetic retinopathy and concomitant microalbuminuria showed typical diabetic glomerulosclerosis and..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.04.007
4
T. Moriya et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
3.2. Morphometric analysis, renal histological injury patterns and renal structural–functional relationships MGV, GBM width, Sv(PGBM/glom), and total Mes/glom were not different between the two groups (Table 1). The values of Vv(Mes/glom) in DR+ were significantly larger than that in DR–. Filtration S/G in DR+ was significantly smaller than that in DR–. The histological injury patterns were significantly different between the two groups. Proportion of C2 in DR+ was significantly increased compared to DR–. We subsequently compared the renal structural–functional relationships according to the presence or absence of DR. Vv(Mes/glom) was significantly and positively correlated with the UAE at biopsy among the patients with DR (Fig. 1). In addition, the histological pattern was not related to the albuminuria category. 3.3. Follow-up data of renal function in typical histological injury pattern and stepwise regression analysis Previously, we indicated that typical diabetic glomerular injury pattern (C2) showed progressive urinary albumin increase and renal function decline (Moriya et al., 2014). In addition, patients with concomitant microalbuminuria and DR showed progressive renal function decline (Moriya et al., 2013). However, it is still unknown whether or not the C2 pattern, the presence of DR and microalbuminuria are related and which combinations affect renal function decline. Therefore, we analyzed the course of renal function in normoalbuminuric and microalbuminuric patients with the C2 pattern and DR (Table 2). Noteworthy, all of the normoalbuminuric patients and 7 of 8 microalbuminuric patients with the C2 pattern had DR among all the patients (Table 2). There were no significant differences in any of the morphometric data between the two groups (Table 2), and baseline GFR did not differ between the two groups. After 7.1 ± 3.8 years of follow-up, UAE did not change from baseline to follow-up in either group. However, GFR in microalbuminuria with C2 had significantly decreased compared to the baseline, while GFR did not change from baseline to follow-up in normoalbuminuria with the C2 pattern (Table 2). Additionally, baseline GFR and UAE in either group did not have any significant differences compared to normoalbuminuria or microalbuminuria with histological patterns out of C2 (data not shown). On the other hand, the patients with DR, but not with C2 pattern (n = 12) and patients without both C2 pattern and DR (n = 6), showed no significant changes of UAE and GFR between the baseline and the follow-up. Vv(Mes/glom) in these 2 groups (0.21 ± 0.03, 0.21 ± 0.05, respectively) and total Mes/glom (0.72 ± 0.19, 0.51 ± 0.11, respectively) were significantly smaller than both normoalbuminuric and microalbuminuric patients with C2 pattern. We subsequently tested what factors, such as mesangial expansion, DR, albuminuria or histological injury pattern mostly
Table 3 Stepwise regression analysis. Independent variables
F-value
P-value
(Dependent variable: Final GFR) Vv(Mes/glom) DR only Microalbuminuria only Histological injury pattern only DR and microalbuminuria
3.360 0.708 2.766 1.934 7.240
0.08 0.41 0.11 0.18 0.01
GFR, glomerular filtration rate; Vv(Mes/glom), volume fraction of mesangium; DR, diabetic retinopathy.
affected the follow-up GFR. The categorization using both DR and microalbuminuria mostly affected GFR at the final observation (Table 3). The categorization of the presence of only DR, or only microalbuminuria, or mesangial expansion, did not significantly affect the final GFR. 4. Discussion We have shown in patients with type 2 diabetes and DR that typical diabetic glomerulosclerosis was significantly present, and GFR decline occurred when they had concomitant microalbuminuria. The study procedures allowed the analyses of structural–functional relationships and renal functional outcomes. It was already shown that renal function loss strikingly occurred in patients with type 2 diabetes with both DR and microalbuminuria using a large number of Japanese patients (Moriya et al., 2013). In Chinese populations, the renal outcome was obviously increased when both DR and microalbuminuria or macroalbuminuria were present (Tong et al., 2007). However, these studies did not show any renal histological evidence in the patients with renal function loss (Moriya et al., 2013; Tong et al., 2007). In terms of relationships between DR and renal histology, the presence of DR has revealed higher incidence of Kimmelstiel–Wilson nodules in patients with type 2 diabetes with hypertension and macroalbuminuria (Schwartz, Lewis, Leonard-Martin, Lewis, & Batlle, 1998). On the other hand, the mesangial volume fraction correlated with DR severity when the patients had hypertension from normoalbuminuria to macroalbuminuria in type 1 diabetes (Chavers, Mauer, Ramsay, & Steffes, 1994). Moreover, renal histological changes were more severe in Caucasian normoalbuminuric patients with type 1 diabetes with DR than those without DR (Klein et al., 2005). However, it remains unknown whether or not DR is related to renal histological changes at the early stage of DN, especially in type 2 diabetes; although one study, utilizing microalbuminuric type 2 diabetes, showed that only patients with typical diabetic glomerulosclerosis had proliferative DR (Fioretto et al., 1996).
Table 2 Follow-up data of renal function between normoalbuminuria and microalbuminuria with typical histological injury patterns and DR.
Number DR UAE at baseline (μg/min) UAE at follow-up (μg/min) P-value (UAE at baseline vs. follow-up) Iohexol-GFR at baseline (ml/min/1.73 m2) Iohexol-GFR at follow-up (ml/min/1.73 m2) P-value (GFR at baseline vs. follow-up) MGV (×106 μm3) GBM width (nm) Vv(Mes/glom) Sv(PGBM/glom) (μm2/μm3) Total Mes/glom (×106 μm3) Filtration S/G (μm2)
Normoalbuminuria with C2
Microalbuminuria with C2
P-value
5 5 5.4 (5.1–14.6) 15.4 (4.6–244.0) 0.834 107.3 ± 19.8 120.6 ± 23.4 0.821 3.2 ± 1.3 810 ± 118 0.31 ± 0.07 0.09 ± 0.03 1.00 ± 0.41 0.26 ± 0.10
7 7 48.6 (28.3–52.0) 71.9 (24.4–428.3) 0.848 130.6 ± 54.7 80.9 ± 42.0 0.048 3.1 ± 0.3 743 ± 117 0.29 ± 0.06 0.09 ± 0.04 0.92 ± 0.21 0.30 ± 0.16
ND ND ND ND 0.925 0.309 0.851 0.470 0.653 0.958 0.695 0.666
Data are n, median (interquartile range) or means ± SD. UAE, urinary albumin excretion; GFR, glomerular filtration rate; ND, not done for selection criterion.
Please cite this article as: Moriya, T., et al., Type 2 diabetic patients with diabetic retinopathy and concomitant microalbuminuria showed typical diabetic glomerulosclerosis and..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.04.007
T. Moriya et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
In the present study, we described the histological injury patterns from normoalbuminuria to microalbuminuria in type 2 diabetes. All microalbuminuric patients with DR, and 5 of 7 normoalbuminuria patients with DR, had the C2 pattern. Furthermore, the patients with DR had significant increases in the mesangial volume fraction as revealed by electron microscopic morphometry compared with that in patients without DR. These data suggested that typical diabetic glomerulosclerosis and renal histological severity related to DR in patients with type 2 diabetes and was compatible with that seen in patients with type 1 diabetes with DR (Klein et al., 2005). Our previous study did not show any correlation between urinary albumin and histological changes including Vv(Mes/glom) at the early stage of DN (Moriya et al., 2002). That study purported that renal structural–functional relationships were thought not to be present at the early stage of DN in patients with type 2 diabetes. However, in the present study, we discovered that there was a positive correlation between the UAE and Vv(Mes/glom) in the patients with DR. This phenomenon suggested that renal structural–functional relationships became evident even at the early stage of DN if the patients had DR. This phenomenon might depend on the fact that renal histological changes, such as diabetic glomerulosclerosis, were more severe in patients with DR, as evidenced in the present study and in another previous study (Klein et al., 2005). More recently, a link between the quantitative assessment of the retinal vessel caliber and the change in the glomerulopathy index, including mesangial expansion and GBM thickening during 5 years of follow-up, was reported in normotensive normoalbuminuric Caucasian type 1 diabetic patients (Klein et al., 2010). Change in the retinal vessel diameter has been speculated to reflect inflammation, endothelial dysfunction, and prevalence and incidence of DR (Nguyen & Wong, 2009). Therefore, DR might reflect renal histological severity as diabetic glomerulosclerosis regardless of albuminuria. We can interpret that the patients without DR make the renal structural– functional relationships complicated in patients with type 2 diabetes. We need to examine other renal histological changes than glomerular changes including tubulointerstitial and vascular damage in these types of cases. In renal function, there were no significant differences of GFR measured by iohexol injection between normoalbuminuria with C2 and microalbuminuria with C2 at the baseline. GFR in microalbuminuria with C2 significantly decreased at the follow-up compared to the baseline. This phenomenon might show that typical diabetic glomerulosclerosis resulted in a GFR decline over several years of follow-up through microalbuminuria, which is compatible with our previous study (Moriya et al., 2014). On the other hand, we have shown that histological severity, not related to initial UAE, predicted an increase in UAE 5–6 years later (Moriya et al., 2008). However, Vv(Mes/glom) itself did not affect the GFR at the follow-up in the present study, while in microalbuminuric Caucasian type 1 diabetic patients, the rate of annual GFR decline was related to the renal histological change (Rudberg & Osterby, 1997). Moreover, the classifications using only microalbuminuria or only DR did not affect the final GFR. Therefore, the present analysis might indicate the consideration that a change in both albuminuria and DR predicts a GFR decline, which is compatible with the previous JDCS (Moriya et al., 2013) and suggests the necessity to refer all such patients to ophthalmologists, especially when they are in the microalbuminuric stage. The limitation of this study was that the relatively small population of patients. Research-related renal biopsies can almost never be performed in our country at the early stage of DN. However, it was evidenced that DR was related to typical histological injury patterns and predict renal function loss when patients had microalbuminuria, as shown in the JDCS (Moriya et al., 2013) using renal biopsy together with the more accurate iohexol-GFR method. Further follow-up study is warranted to examine a larger population of
5
patients over a longer period of time. Another limitation was that there might be selection bias of the patients because there were no hypertensive patients at the baseline. Hypertension is known to have effects on retinopathy since long-term elevation of BP has been shown to be associated to retinopathy (Avery et al., 2012). It has also been reported that the severity of DR and nephropathy were related in the presence of hypertension (Chavers et al., 1994). However, the present study focused on the early stage of diabetic nephropathy. In addition, we wanted to avoid the effect of BP on the renal function course because we planned to test the effect of DR itself on renal function. Further study is warranted to examine the effects of BP on DR in Japanese type 2 diabetic patients. In summary, in the normo- and microalbuminuric patients with type 2 diabetes with a definite histological diagnosis of DN, renal structural–functional relationships became more obvious in the presence of DR, and microalbuminuria with DR showed typical diabetic glomerulosclerosis and development of GFR decline. Therefore, both DR and microalbuminuria should be considered to determine whether or not the renal function progresses in patients with type 2 diabetes.
Acknowledgements The authors thank Eriko Oda and Noriko Nemoto of the Kitasato Bio-Imaging Center for their technical assistance. The study was presented in part at the 71st Annual Scientific Meeting of the American Diabetes Association, 2011. We also thank Robert E. Brandt, Founder, CEO, and CME, of MedEd Japan, for editing and formatting the manuscript.
References Avery, C. L., Kucharska-Newton, A., Monda, K. L., Richy Sharrett, A., Mosley, T. H., Klein, B. E., ... Klein, R. (2012). Impact of long-term measures of glucose and blood pressure on the retinal microvasculature. Atherosclerosis, 225, 412–417. Chavers, B. M., Bilous, R. W., Ellis, E. N., Steffes, M. W., & Mauer, S. M. (1989). Glomerular lesions and urinary albumin excretion in type I diabetes without overt proteinuria. The New England Journal of Medicine, 320, 966–970. Chavers, B. M., Mauer, S. M., Ramsay, R. C., & Steffes, M. W. (1994). Relationship between retinal and glomerular lesions in IDDM patients. Diabetes, 43, 441–446. Committee of the Japan Society on the Diagnostic Criteria of Diabetes Mellitus, Seino, Y., Nanjo, K., Tajima, N., Kadowaki, T., Kashiwagi, A., Araki, E., et al. (2010). Report of the committee on the classification and diagnostic criteria of diabetes mellitus. Journal of Diabetes Investigation, 1, 212–228. Fioretto, P., Mauer, M., Brocco, E., Velussi, M., Frigato, F., Muollo, B., ... Nosadini, R. (1996). Patterns of renal injury in NIDDM patients with microalbuminuria. Diabetologia, 39, 1569–1576. Fioretto, P., Steffes, M. W., & Mauer, M. (1994). Glomerular structure in nonproteinuric IDDM patients with various levels of albuminuria. Diabetes, 43, 1358–1364. Jensen, E. B., Gundersen, H. J., & Osterby, R. (1979). Determination of membrane thickness distribution from orthogonal intercepts. Journal of Microscopy, 115, 19–33. Jin, Y., Moriya, T., Tanaka, K., Matsubara, M., & Fujita, Y. (2006). Glomerular hyperfiltration in non-proteinuric and non-hypertensive Japanese type 2 diabetic patients. Diabetes Research and Clinical Practice, 71, 264–271. Kanauchi, M., Kawano, T., Uyama, H., Shiiki, H., & Dohi, K. (1998). Discordance between retinopathy and nephropathy in type 2 diabetes. Nephron, 80, 171–174. Klein, R., Knudtson, M. D., Klein, B. E., Zinman, B., Gardiner, R., Suissa, S., ... Mauer, M. (2010). The relationship of retinal vessel diameter to changes in diabetic nephropathy structural variables in patients with type 1 diabetes. Diabetologia, 53, 1638–1646. Klein, R., Zinman, B., Gardiner, R., Suissa, S., Donnelly, S. M., Sinaiko, A. R., ... Mauer, M. Renin-Angiotensin System Study. (2005). The relationship of diabetic retinopathy to preclinical diabetic glomerulopathy lesions in type 1 diabetic patients: The Renin-Angiotensin System Study. Diabetes, 54, 527–533. Krutzen, E., Back, S. E., Nilsson-Ehle, I., & Nilsson-Ehle, P. (1984). Plasma clearance of a new contrast agent, iohexol: A method for the assessment of glomerular filtration rate. The Journal of Laboratory and Clinical Medicine, 104, 955–961. Mauer, S. M., Steffes, M. W., Ellis, E. N., Sutherland, D. E., Brown, D. M., & Goetz, F. C. (1984). Structural-functional relationships in diabetic nephropathy. The Journal of Clinical Investigation, 74, 1143–1155. Mauer, M., Zinman, B., Gardiner, R., Suissa, S., Sinaiko, A., Strand, T., ... Klein, R. (2009). Renal and retinal effects of enalapril and losartan in type 1 diabetes. The New England Journal of Medicine, 361, 40–51.
Please cite this article as: Moriya, T., et al., Type 2 diabetic patients with diabetic retinopathy and concomitant microalbuminuria showed typical diabetic glomerulosclerosis and..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.04.007
6
T. Moriya et al. / Journal of Diabetes and Its Complications xxx (2016) xxx–xxx
Moriya, T., Moriya, R., Yajima, Y., Steffes, M. W., & Mauer, M. (2002). Urinary albumin as an indicator of diabetic nephropathy lesions in Japanese type 2 diabetic patients. Nephron, 91, 292–299. Moriya, T., Suzuki, Y., Inomata, S., Iwano, M., Kanauchi, M., & Haneda, M. (2014). Renal histological heterogeneity and functional progress in normoalbuminuric and microalbuminuric Japanese patients with type 2 diabetes. BMJ Open Diabetes Research & Care, 2, e000029. Moriya, T., Tanaka, K., Hosaka, T., Hirasawa, Y., & Fujita, Y. (2008). Renal structure as an indicator for development of albuminuria in normo- and microalbuminuric type 2 diabetic patients. Diabetes Research and Clinical Practice, 82, 298–304. Moriya, T., Tanaka, S., Kawasaki, R., Ohashi, Y., Akanuma, Y., Yamada, N., ... Katayama, S. (2013). Japan Diabetes Complications Study Group. Diabetic retinopathy and microalbuminuria can predict macroalbuminuria and renal function decline in Japanese type 2 diabetic patients: Japan Diabetes Complications Study. Diabetes Care, 36, 2803–2809. Moriya, T., Tsuchiya, A., Okizaki, S., Hayashi, A., Tanaka, K., & Shichiri, M. (2012). Glomerular hyperfiltration and increased glomerular filtration surface are associated with renal function decline in normo- and microalbuminuric type 2 diabetes. Kidney International, 81, 486–493. Nguyen, T. T., & Wong, T. Y. (2009). Retinal vascular changes and diabetic retinopathy. Current Diabetes Reports, 9, 277–283.
Rudberg, S., & Osterby, R. (1997). Decreasing glomerular filtration rate–An indicator of more advanced diabetic glomerulopathy in the early course of microalbuminuria in IDDM adolescents? Nephrology, Dialysis, Transplantation, 12, 1149–1154. Schwartz, M. M., Lewis, E. J., Leonard-Martin, T., Lewis, J. B., & Batlle, D. (1998). Renal pathology patterns in type II diabetes mellitus: Relationship with retinopathy. The Collaborative Study Group. Nephrology, Dialysis, Transplantation, 13, 2547–2552. Tong, P. C., Kong, A. P., So, W. Y., Yang, X., Ng, M. C., Ho, C. S., ... Cockram, C. S. (2007). Interactive effect of retinopathy and macroalbuminuria on all-cause mortality, cardiovascular and renal end points in Chinese patients with type 2 diabetes mellitus. Diabetic Medicine, 24, 741–746. Trevisan, R., Vedovato, M., Mazzon, C., Coracina, A., Iori, E., Tiengo, A., ... Del Prato, S. (2002). Concomitance of diabetic retinopathy and proteinuria accelerates the rate of decline of kidney function in type 2 diabetic patients. Diabetes Care, 25, 2026–2031. Weibel, E. R. (1979). Stereological methods. In E. R. Weibel (Ed.), Practical methods for biological morphometry, Vol 1. (pp. 9–62). London: Academic Press. Wilkinson, C. P., Ferris, F. L., III, Klein, R. E., Lee, P. P., Agardh, C. D., Davis, M., ... Verdaguer, J. T.Global Diabetic Retinopathy Project Group. (2003). Proposed international clinical diabetic retinopathy and diabetic macular edema disease severity scales. Ophthalmology, 110, 1677–1682.
Please cite this article as: Moriya, T., et al., Type 2 diabetic patients with diabetic retinopathy and concomitant microalbuminuria showed typical diabetic glomerulosclerosis and..., Journal of Diabetes and Its Complications (2016), http://dx.doi.org/10.1016/j.jdiacomp.2016.04.007