The relationship of retinal vessel diameter to changes in diabetic nephropathy structural variables in patients with type 1 diabetes
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We examined whether retinal vessel diameter in persons with type 1 diabetes mellitus is associated with changes in subclinical anatomical and functional indicators of diabetic nephropathy.
Persons with type 1 diabetes mellitus had gradable fundus photographs and renal biopsy data at baseline and 5-year follow-up (n = 234). Retinal arteriolar and venular diameters were measured at baseline and follow-up. Central retinal arteriole equivalent (CRAE) and central retinal venule equivalent (CRVE) were computed. Baseline and 5-year follow-up renal structural variables were assessed by masked electron microscopic morphometric analyses from percutaneous renal biopsy specimens. Variables assessed included: mesangial fractional volume, glomerular basement membrane width, mesangial matrix fractional volume and glomerular basement membrane width composite glomerulopathy index.
While controlling for other covariates, baseline CRAE was positively associated with change in the glomerulopathy index over the 5-year period. Change in CRAE was inversely related to a change in mesangial matrix fractional volume and abnormal mesangial matrix fractional volume, while change in CRVE was directly related to change in the volume fraction of cortex that was interstitium [Vv(Int/cortex)] over the 5-year period. Baseline CRAE or CRVE or changes in these diameters were not related to changes in other anatomical or functional renal endpoints.
Independently of other factors, baseline CRAE correlated with changes in glomerulopathy index, a composite measure of extracellular matrix accumulation in the mesangium and glomerular basement membrane. A narrowing of the CRAE was related to mesangial matrix accumulation. Changes in CRVE were related to changes in Vv(Int/cortex), a measure of interstitial expansion in persons with type 1 diabetes mellitus.
KeywordsEpidemiology Microvascular disease Nephropathy Retinal blood vessel diameters Risk indicators Type 1 diabetes mellitus
Central retinal arteriole equivalent
Central retinal venule equivalent
Early Treatment Diabetic Retinopathy Study
Glomerular basement membrane
Index of arteriolar hyalinosis
Renin–Angiotensin System Study
Surface density (surface/volume) of peripheral glomerular capillary glomerular basement membrane per glomerulus
Volume fraction of cortex that was interstitium
Mesangial fractional volume per glomerulus
Mesangial matrix fractional volume per glomerulus
Wisconsin Epidemiologic Study of Diabetic Retinopathy
Description of cohort
The RASS was a parallel, double-blind, placebo-controlled, multi-centre clinical trial of diabetic nephropathy primary prevention and retinopathy development and progression conducted at three clinical centres in Minneapolis (MN, USA), Montreal (QC, Canada) and Toronto (ON, Canada). The study design and cohort description have been detailed elsewhere . All data were collected with Institutional Review Board approval in conformity with all federal, state and provincial laws, and the study was in adherence with the tenets of the Declaration of Helsinki as revised in 1983. Informed consent was obtained. Participants were 15 years of age or older, with type 1 diabetes duration of 2–20 years and disease onset before the 45th birthday. All participants were normotensive (BP <135/85 mmHg), normoalbuminuric (AER < 20 μg/min on at least two of three timed overnight urine collections) and had normal or increased GFR (≥90 ml min−1 1.73 m−2). Participants (n = 285) were randomised into one of the following three treatment groups: losartan (angiotensin-receptor blocker), enalapril (ACE inhibitor) or placebo .
Blood pressure, weight, height and retinal measurements
The baseline examination included measurement of BP in a seated position after resting for 5 min, using an automated device (DinaMap Monitor 18465X; Critikon, Palatine, IL, USA). Height and weight were measured according to standard anthropometric procedures. Pupils were dilated and 30° colour stereoscopic fundus photographs were taken of the seven standard fields as defined in the Early Treatment Diabetic Retinopathy Study (ETDRS) protocol . The photographs were graded in a masked fashion by the University of Wisconsin Ocular Epidemiology Reading Center using the modified Airlie House classification scheme and the ETDRS retinopathy severity scale. Grading protocols and retinopathy severity scales have been described in detail elsewhere [14, 15]. Diabetic retinopathy severity, based on the more severe eye, was grouped as follows: none (level 10), mild diabetic retinopathy (levels 20–43) and moderate to severe diabetic retinopathy (levels ≥47).
Retinal vessel diameters were measured at baseline and at the 5-year follow-up RASS examinations using a computer-assisted technique based on a standard protocol, according to which retinal photographs of field 1 (centred at the optic nerve head) were converted to digital images by a high-resolution scanner using identical settings for all photographs . Retinal vessel measurements were done independently for each examination and each eye. Trained graders who were masked to participant characteristics measured the diameters of all arterioles and venules coursing through a specified area of a half- to a whole disc diameter surrounding the optic disc by means of a software program (University of Wisconsin–Madison, Madison, WI, USA). On average, between 7 and 14 arterioles and an equal number of venules were measured per eye. Individual arteriolar and venular measurements were combined into summary indices reflecting the average retinal arteriolar, i.e. central retinal arteriole equivalent (CRAE), and retinal venular, i.e. central retinal venule equivalent (CRVE), diameter of an eye based on the Parr–Hubbard–Knudtson formula . Graders regularly participated in quality control exercises; the inter- and intra-grader variability was small (interclass and intraclass correlations >0.90 for CRAE and CRVE).
Measures of renal function and glycaemia
GFR was estimated at baseline and follow-up by plasma disappearance of non-radioactive iohexol (Omnipaque 300; Shearing Plough Welwyn Garden City, UK) as detailed elsewhere and expressed in millilitres per minute adjusted up to 1.73 m2 [18, 19]. AER was measured in timed overnight urine collections using a sensitive fluorescence immunoassay  and expressed in micrograms per minute. Normoalbuminuria was defined as having at least two of three AER readings <20 µg/min. HbA1c was measured by HPLC (Diamat HbA1c analyser; Bio-Rad, Hercules, CA, USA) until April 2002, when the Tosoh method was introduced (Tosoh Medics, San Francisco, CA, USA) .
Percutaneous renal biopsies were obtained within 6 months prior to baseline and at follow-up 5 years after randomisation . Tissue processing was as described elsewhere . The centre-most intact glomeruli in the electron microscopy blocks were selected for study and an image calibration grid was obtained at the beginning of each microscopy session . Electron microscopy digital images (magnification ×3,900) stored in tagged image file format (Photoshop 6.0; Adobe, San Jose, CA, USA) were used to build montages of entire glomerular cross-sections, on which counting grids were superimposed. These montages were used to measure the fraction of the glomerulus occupied by the mesangium or the mesangial fractional volume per glomerulus (Vv[Mes/glom]), the mesangial matrix fractional volume per glomerulus (Vv[MM/glom]), the mesangial cell fractional volume and the surface density (surface/volume) of peripheral glomerular capillary glomerular basement membrane (GBM) per glomerulus (Sv[PGBM/glom]) [8, 10, 23, 24, 25, 26, 27]. After the centre-most glomerulus in a block was randomly entered, 10–20 evenly spaced digital images representing an unbiased systematic sample of approximately 30% of the glomerular profile were obtained at magnification ×11,000 for measurement of GBM width [8, 23, 25, 26] and mesangial composition [8, 10, 27].
A glomerulopathy index, expressed as GBM width/10 + [Vv(MM/glom) × 100] and representing a composite expression of glomerular extracellular matrix accumulation in diabetes, was computed using the formula as described by Rudberg et al. . All electron microscopy measurements were done by a single masked observer.
Zenker’s fixed periodic acid–Schiff’s stained light microscopy slides were randomly selected to determine the index of arteriolar hyalinosis (IAH) in all cortical arterioles ≤1 average tubular diameter in size, based on the fraction of each arteriolar wall replaced by hyaline as simultaneously estimated by two masked observers. IAH was calculated as published . Volume fraction of cortex that was interstitium (Vv[Int/cortex]) and volume fraction of cortical tubules per total tubules that were atrophic were estimated by a masked observer at magnification ×300 by point counting .
Statistical analyses were conducted in SAS version 9 (SAS Institute, Cary, NC, USA). Means were compared for statistically significant differences by the t test and ANOVA when two or more than two groups, respectively, were involved. Pearson correlation coefficients were computed for pairwise associations of variables. Multivariable associations between anatomical and functional renal endpoints (and changes in these measures), and CRAE, CRVE and other independent variables such as age, sex, duration of diabetes, HbA1c, BP and body mass were explored by multiple linear regression. Similarly, multivariable relationships between anatomical and functional renal characteristics defined as normal or abnormal, and CRAE or CRVE and other independent variables were evaluated by logistic regression, with selected covariates in the model chosen because they were statistically significantly associated with the specific outcome.
Characteristics of the cohort baseline examination
Of the 285 participants, one had insufficient glomeruli for renal morphometric measurements, 28 had renal biopsy data without gradable fundus photographs for retinal vessel measurement and 257 had gradable fundus photographs and biopsy data at baseline. Persons with gradable fundus photographs and biopsy data were older than those without gradable fundus photographs and biopsy data (p = 0.002); there were no statistically significant differences between groups for other characteristics .
Baseline characteristics of the RASS cohort
Mean (SD) or %
Male sex (%)
Diabetes duration (years)
Body mass index (kg/m2)
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Mean arterial blood pressure (mmHg)
Serum creatinine (µmol/l)
GFR (ml min−1 1.73 m−2)
Albumin excretion rate (µg/min)
Mesangial cell fractional volume/glomerulusa
GBM widtha (nm)
Volume fraction of cortical tubules per total of atrophic tubulesc
Diabetic retinopathy severity
Moderate to severe (%)
Participants, total: n = 257
Relationships between both CRAE and CRVE and change in renal endpoints
Of the 257 participants with gradable retinal vessel measurements and biopsy data at baseline, 234 had gradable fundus photographs and biopsy data at follow-up. Persons with biopsy data at follow-up were younger than those without (p = 0.03); additionally, persons without follow-up biopsy data and gradable fundus photographs had larger baseline Vv(Mes/glom) (p = 0.04) and Vv(MM/glom) (p = 0.002) than those with biopsy data. Otherwise, there were no statistically significant differences between groups for other characteristics .
Correlations of CRAE and CRVE at baseline with change in diabetic nephropathy measures and incident abnormal values over 5-year follow-up
Participants at risk, na
GBM width (nm)
Vv(Mes/glom) (n = 30)
Vv(MM/glom) (n = 43)
Vv(MC/glom)c (n = 20)
GBM width (n = 27)
Sv(PGBM/glom) (n = 61)
Glomerulopathy index (n = 27)
Relation of CRAE and CRVE at baseline to change in renal endpoint models
CRAE per SD
CRVE per SD
Change in GFR
Change in AER
Change in Vv(Mes/glom)
Change in Vv(MM/glom)
Change in Vv(MC/glom)b
Change in Vv(Int/cortex)
Change in Vv(AT/TT)c
Change in GBM width (nm)
Change in Sv(PGBM/glom)
Change in glomerulopathy index
Relation of CRAE and CRVE at baseline to 5-year incidence of renal abnormality models
CRAE per SD
CRVE per SD
OR (95% CI)b
OR (95% CI)b
OR (95% CI)b
OR (95% CI)b
Abnormal GBM width
While controlling for duration of diabetes, CRVE was positively associated with change in Vv(MM/glom), glomerulopathy index and Sv(PGBM/glom), and with the incidence of abnormal Sv(PGBM/glom) and abnormal glomerulopathy index (Tables 3 and 4). None of these relationships remained statistically significant while controlling for other covariates in the model.
Relationships between changes in both CRAE and CRVE, and changes in renal endpoints
Changes in CRAE over the 5-year period were related to changes in Vv(MM/glom) (r = −0.14, p = 0.04) and incident abnormal Vv(MM/glom) (r = −0.17, p = 0.03), while changes in CRVE were related to changes in Vv(Int/cortex) (r = 0.20, p < 0.01). The relation of changes in CRAE remained associated with changes in Vv(MM/glom), incident abnormal Vv(MM/glom) and the relation between changes in CRVE and changes in Vv(Int/cortex), while controlling for other risk factors (data not shown). Changes in CRAE or CRVE were not statistically significantly associated with changes in other renal variables (data not shown).
Changes in retinal vessel diameters occur early in the course of type 1 diabetes mellitus, possibly reflecting structural and/or functional alterations resulting from changes in retinal blood flow and the effects of chronic hyperglycaemia, inflammation and endothelial dysfunction [30, 31]. Retinal arteriolar narrowing (a small CRAE), for example, is thought to be related to cumulative small vessel damage from the effects of hypertension, while retinal venular widening (a large CRVE) has been thought to reflect retinal hyperperfusion and/or lactic acidosis associated with hyperglycaemia and hypoxia (Fig. 2) [16, 30, 31]. In persons with type 1 diabetes mellitus, narrower retinal arteriolar and wider retinal venular diameters are thought to reflect microvascular changes from processes described above independently of diabetic retinopathy severity [32, 33, 34, 35, 36, 37, 38, 39]. These may be markers of similar changes in the microvascular diameters in the cerebral, coronary, peripheral and renal circulations in persons with or without type 1 diabetes mellitus; they may also be indicators of pathogenetic processes that are damaging to other targets of diabetic microvascular injury [33, 39, 40].
The RASS is the first study to examine the relationship of CRAE and CRVE with changes in diabetic renal structural variables in sequential research biopsies in patients with type 1 diabetes mellitus. CRAE and CRVE were associated with abnormalities and increases in renal structural variables that reflect extracellular matrix accumulation, the central pathological processes leading to renal dysfunction in diabetic nephropathy [7, 9]. These abnormalities included: (1) increases in GBM width and mesangial matrix fractional volume, which, as with the composite glomerulopathy index, are closely associated with albuminuria in type 1 diabetes mellitus; and (2) decrease in glomerular filtration surface density Sv(PGBM/glom), which results from mesangial expansion ; and they are ultimately associated with GFR loss in type 1 diabetes mellitus (Fig. 1) .
In the main, these relationships were independent of diabetes duration or severity of diabetic retinopathy. Speculation on these associations could take several directions. Increased CRVE and CRAE could be related to increased retinal blood flow, possibly mirrored by increases in renal blood flow and long posited as a candidate mechanism for diabetic nephropathy pathogenesis through haemodynamic injury . Alternatively, increased retinal and renal blood flow could reflect compensatory responses to metabolic disturbances that could be the drivers of tissue injury in diabetes, e.g. relative tissue hypoxia  or oxidative stress .
Regardless of the mechanism, the RASS, which directly compares structural changes at relatively early stages of diabetic nephropathy and diabetic retinopathy, provides additional information, which indicates important relationships between these two microvascular complications and could not be derived from renal functional studies, since these were all normal at RASS baseline. It is not clear from these data whether the associations between baseline retinal diameter variables and the change in important structural variables of diabetic nephropathy in type 1 diabetes mellitus patients without renal functional abnormalities will actually make early diabetic nephropathy risk predictions more precise . In contrast to the relationship between baseline CRAE and renal structural change as measured by glomerulopathy index, an increase in CRAE over the 5 years of RASS was inversely related to Vv(MM/glom). Clearly, longer term studies involving larger numbers of type 1 diabetic patients with hypertension and albuminuria would be necessary to further describe and unravel these complex dynamic relationships.
In the RASS, an increase in CRVE was related to an increase in Vv(Int/cortex). The latter is a measure of interstitial expansion, which at these earlier stages of diabetic nephropathy would mainly result from an increase in the cellular component of the interstitium . Wider retinal venules are associated with systemic inflammation, consistent with the finding that the latter may be involved in the early increase in Vv(Int/cortex) .
No relationships were found in the RASS between retinal vessel diameters and renal functional endpoints. This was in contrast to findings in type 1 diabetes mellitus patients in the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR) . In that study, the risk of incident proteinuria and renal insufficiency was increased (RR for 4th vs 1st to 3rd quartiles 1.53, 95% CI 1.19–1.97 and 1.41, 1.05–2.17 respectively) in persons with wider retinal venular diameters, the increase being independent of age, sex, duration of diabetes, HbA1c, baseline diabetic retinopathy severity and other factors. No relationship was found between retinal arteriolar diameter at baseline and these renal endpoints in that study. Comparisons of RASS findings with those from the WESDR are limited due to differences in the two cohorts. Thus the WESDR reported on functional outcomes in a diabetic cohort including patients with hypertension and microalbuminuria at baseline, whereas the RASS cohort members were all normotensive and normoalbuminuric at baseline. Moreover, WESDR participants were studied in a period of poorer glycaemic control. It can only be conjectured that if the RASS (like the WESDR) had included hypertensive albuminuric patients with longer duration of type 1 diabetes, it might have found stronger associations between retinal vessel diameters and anatomical renal abnormalities.
Our study has a number of strengths, including the objective determination of retinal vessel calibre using standardised protocols and the use of careful morphometric techniques in two research renal biopsies spaced 5 years apart. However, caution should be exercised in interpreting the findings. It is possible that the angiotensin-receptor blocker or ACE inhibitor treatment used in the RASS masked a possible relationship between retinal vessel measurements and renal endpoints. Although neither treatment arm in our study was shown to have a protective effect on structural or functional renal endpoints , angiotensin-receptor blocker and ACE inhibitor treatment significantly reduced retinopathy progression in the RASS. This may have limited detection of associations of changes in CRAE or CRVE with renal structural or functional endpoints, which might have been present in a natural history study. However, when analyses were limited to the 85 persons in the placebo group the associations of CRAE or CRVE with the renal endpoints were essentially unchanged (R. Klein, unpublished data). In addition, our study included estimates of CRAE and CRVE taken at a single and random point in the pulse cycle. However, we have previously shown that the effect of variability from the pulse cycle was not large and also not likely to affect any of the associations under study . It is possible that the choice of CRAE as marker of narrowing of retinal arterioles may not have been as sensitive as other measures of structural and physiological changes in the microvasculature, e.g. retinal arteriole wall-to-lumen ratio; this could have diminished our ability to demonstrate stronger relationships with renal structural abnormalities .
In summary, retinal vessel diameters were related to the early stages of diabetic nephropathy lesion development in terms of matrix accumulation and interstitial expansion, but were unrelated to changes in renal function in normotensive, normoalbuminuric persons with type 1 diabetes mellitus. At this point in time, we can only speculate that widened retinal venular diameter reflects the cumulative effects of various diabetic processes on the systemic microvasculature, including the kidney. However, it is too early to use this measure clinically to determine risk of renal anatomical damage prior to the appearance of functional changes, e.g. albuminuria and decreasing glomerular filtration, in people with type 1 diabetes.
This study was funded by research grants from the Juvenile Diabetes Research Foundation (R. Klein) and the National Institutes of Health (NIH), National Institute of Diabetes and Digestive and Kidney Disease (NIH DK51975). Work was also supported by Merck (in the USA), Merck Frosst (in Canada) and the Canadian Institutes of Health Research (CIHR) (DCT 14281). The University of Minnesota General Clinical Research Center is supported by the NIH (M01 RR 00400). Additional support was given by the National Institutes of Health, National Eye Institute (EY12198) and Research to Prevent Blindness (Senior Scientific Investigator Award to R. Klein) (New York, USA). S. Suissa was the recipient of a Distinguished Investigator Award from the CIHR. The sponsors and funding organisations had no role in the design or conduct of this research.
Duality of interest
B. Zinman has received lecture fees, consulting fees and research grants from Merck. R. Klein is an Advisory Board member for AstraZeneca (through the DIRECT study), Pfizer, Lilly and Novartis. All other authors declare that there is no duality of interest associated with this manuscript.
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