Nephrol Dial Transplant (2001) 16: 2337–2347
Original Article
Renal function and structure in albuminuric type 2 diabetic patients
without retinopathy
Per Knud Christensen1, Svend Larsen 2, Thomas Horn2, Steen Olsen 2,
and Hans-Henrik Parving1
1
Steno Diabetes Center, Gentofte and 2 Department of Pathology, Herlev Hospital, Herlev, Denmark
Abstract
Background. In type 2 diabetic patients without
retinopathy the cause of albuminuria is heterogeneous
and our knowledge of the relationship between kidney
structure and function in these patients is limited.
Therefore, a long-term study evaluating the structural–
functional relationship in albuminuric type 2 diabetic
patients without retinopathy was performed.
Methods. Mesangial volume of total glomerular
volume (Vv (mesuglom)), fractional area of focal
interstitial fibrosis and tubular atrophy of cortical
area (FF) and percentage of sclerosed glomeruli
(SuG) were measured on kidney biopsies from 49 type
2 diabetic patients without retinopathy. Glomerular
filtration rate (GFR) was determined at least 3 times
(median 8 (range 3–20)) in each patient. Patients were
followed for 7.0 (1.1–17) years. Albuminuria and
blood pressure were measured every 3–6 months.
Results. Biopsies revealed diabetic glomerulopathy
(DG-group) in 69% of the patients (27 maleu7 female)
and normal glomerular structure (n s9) or glomerulonephritis (ns 6) were found in 31% (13 maleu2 female)
(NDG-group). In the DG-group GFR decreased
from 97"5 to 66"5 mluminu1.73 m2 (mean"SE)
(P-0.001), with a rate of decline in GFR of
5.3"0.8 mluminuyear and in the NDG-group from
93"7 to 74"11 mluminu1.73 m2 (P-0.01), with
a rate of decline in GFR of 3.2"0.9 mluminuyear,
P s0.09 between groups. Mean arterial blood pressure
decreased from 109"2 to 100"2 mm Hg (P-0.001)
(DG-group) and remained unchanged in the NDGgroup. An association between Vv (mesuglom) and rate
of decline in GFR was revealed mainly in the NDGgroup (DG-group; r s0.31, P s 0.07 and NDG-group;
rs 0.74, P-0.01). Furthermore, the rate of decline in
GFR seemed to be associated with FF in the NDG
group (rs 0.48, P s 0.07). Percentage of SuG was not
Correspondence and offprint requests to: Per K. Christensen, Steno
Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte,
Denmark. Email: [email protected]
#
associated with the rate of decline in GFR.
Vv (mesuglom) was associated with mean albuminuria
during follow-up in the DG group; rs 0.38, P-0.03
(NDG group; rs 0.51, P s 0.09). Albuminuria was
an independent predictor of the rate of decline in GFR
in both groups (DG-group; rs 0.40, P-0.05 and
NDG-group; rs 0.61, P-0.01).
Conclusions. Our study revealed a tendency to a faster
rate of decline in GFR in the DG-group compared
to the much smaller NDG-group, characterized by
marked heterogeneity of the underlying kidney lesions
and rate of GFR loss. A large mesangial volume
fraction was associated with increased albuminuria
and loss in GFR. Albuminuria acted as a progression
promoter in both groups.
Keywords: albuminuria; diabetic glomerulopathy;
glomerular filtration rate; hypertension; mesangium;
non-diabetic glomerulopathies; retinopathy; type 2
diabetes mellitus
Introduction
In both type 1 and type 2 diabetic patients with
retinopathy the development of persistent albuminuria
)300 mgu24 h is the major criterion for the clinical
diagnosis of diabetic nephropathy w1,2x. However,
biopsy studies have revealed a prevalence of nondiabetic kidney disease in 5% of albuminuric type 1
diabetic patients w1x as compared with 10–24% in
albuminuric type 2 diabetic patients w2,3x, and an even
higher prevalence (31%) in type 2 diabetic patients
without retinopathy w4x. Despite the heterogeneous
nature of the underlying causes of albuminuria in
patients with type 2 diabetes, and an increasing prevalence of end-stage renal disease among these patients,
most studies of renal morphology in diabetes have
mainly concentrated on type 1 diabetes. Only a
relatively few long-term studies have dealt with the
relationship between kidney structure and function
2001 European Renal Association–European Dialysis and Transplant Association
2338
in albuminuric type 2 diabetic patients w2,5–9x
Originally Østerby et al. w5x found a close correlation
between renal structure and functional changes in
albuminuric type 2 diabetic patients. Furthermore,
a correlation between severity of retinopathy and
the severity of structural lesions in the kidney was
revealed. The association between retinopathy and
albuminuria underlines the generalized nature of the
diabetic angiopathy. However, the association is not
always complete, since a high percentage (22–62%) of
albuminuric type 2 diabetic patients do not have
diabetic retinopathy. Unfortunately the course of
glomerular filtration rate (GFR) and the structural–
functional relationship in albuminuric type 2 diabetic
patients without retinopathy has only been scantily
investigated.
The aim of the present longitudinal observational
long-term study was to evaluate the clinical course of
GFR in a large group of consecutive albuminuric
type 2 diabetic patients without retinopathy. Secondly,
we evaluated the relationships between the decline
in kidney function and kidney structure, arterial
blood pressure, albuminuria, glycaemic control, lipids,
known duration of diabetes and baseline GFR.
A cross-sectional report on the present patients has
been presented previously w4x.
Subjects and methods
Subjects
Patients were considered to have type 2 diabetes if they
were treated with diet alone, or in combination with oral
hypoglycaemic agents, or if they were treated with insulin
and had an onset of diabetes after the age of 40 years and a
body weight in excess of the ideal body weight at the time of
diagnosis. All insulin-treated patients who were lean at the
time of diagnosis had a glucagon test performed, and type 2
diabetes was diagnosed if a stimulated C-peptide value was
equal to or above 0.60 pmoluml.
All Caucasian (n s 347) patients with type 2 diabetes and
persistent albuminuria ()300 mgu24 h in at least two out of
three consecutive, sterile non-ketotic 24-h urine samples),
who had attended the out patient clinic at Steno Diabetes
Center between 1978 and 1998, were evaluated. Ninety-three
of these patients had no retinopathy. After 1992 our nephrologists decided not to perform kidney biopsy in patients
with type 2 diabetes without retinopathy when albuminuria
was less than 1 gu24 h. Consequently 15 patients without
retinopathy with albuminuria -1 gu24 h were excluded.
Twenty of the remaining 78 patients were not referred for
a kidney biopsy for several reasons; (i) four patients died
shortly after onset of albuminuria, (ii) seven patients were
older than 65 years, and (iii) contraindications were present
in nine patients (four had a solitary kidney, one had contracted kidneys and four patients were treated with anticoagulant medication because of cardiovascular disease). The
rest (n s 58) of the patients were referred for a kidney biopsy,
six of these patients later decided not to participate, and one
kidney biopsy was not successful.
Two of the remaining 51 patients were later excluded: one
because of long-term treatment with acetazolamide and in
P. K. Christensen et al.
one patient it was not possible to estimate the rate of decline
in GFR, because fewer than three GFR measurements were
performed.
All (n s 52) patients gave informed consent and the study
was performed in accordance with the Helsinki declaration.
Methods
The biopsies were evaluated by three masked nephropathologists. To excludeuminimize bias with respect to
presence of diabetic kidney lesions and to increase possible
variability in the morphometric analyses, we mixed biopsies
from diabetic patients with 14 biopsies from patients without
diabetes from our file, who had normal renal function, and
normal structure by light microscopy. All morphometric
measurements were made by one of the authors (S.O.).
Light microscopy
The tissue preparation has been described in detail previously
w4x. Diffuse diabetic glomerulopathy, hyaline arteriolosclerosis, tubular atrophy, and interstitial fibrosis were semiquantitatively estimated. The final reading (0, q1, q2, q3)
was made by agreement of the three nephropathologists
watching together at a video screen attached to the microscope. The number of totally sclerotic glomeruli was counted
and expressed as percentage of the total number.
Morphometry
The mesangial volume fraction (mesangium in fraction of the
total glomerular volume (Vv (mesuglom)) was measured at
light microscopy by point counting using a computer-assisted
stereological system (GRID, Interactivision, DK). Magnification on the video screen was 3 1650. The grid had 5 3 6
points. The number of points hitting the mesangium (matrix
as well as cells) was expressed as the fraction of the points
hitting the reference space, which was the glomerulus defined
as the circumscribed minimal polygon. The measurements
were made on one silver methenamine stained 2 m section
from each biopsy. One field from each of 6–10 glomeruli
was measured (corresponding to 180–300 reference points),
excluding totally or partially sclerosed glomeruli.
Interstitial fibrosis and tubular atrophy always occurred in
sharply defined focal areas. Each fibrotic scar was circumscribed and their areas automatically computed by the
system. The sum of these areas was expressed as the percentage of the total cortical area in the biopsy. In order not
to miss any diffuse interstitial fibrosis outside these focal
areas, we also measured the fibrosis outside the focal areas by
point counting. The interstitial tissue including capillaries
was expressed as a fraction of the cortical labyrinth (cortex
minus glomeruli and vessels larger than capillaries). Tubular
basement membranes were not included in the interstitial
tissue.
Immunofluorescence microscopy
Kidney specimens were frozen using dry ice, embedded
in Tissue-Teck (Miles, Naperville, Illinois, USA) gelatin, and
2 mm sections were cut at 248C on a Leitz Histocryotome
(Wetzlar, Germany). Direct immunofluorescent staining
technique was applied, using FITC-conjugated rabbit or goat
Renal structure and function in diabetic patients
antisera specifically reactive to human IgG, IgM, IgA as well
as complement C1q, C3 and C4.
2339
follow-up GFR was measured 8 times (3–20), albuminuria 29
(4–75), arterial blood pressure 30 (5–90), HbA1c 20 (4–20),
total serum cholesterol 11 (1–30) and serum HDL cholesterol
8 (1–18) times (medians (ranges)).
Laboratory techniques
Retinopathy was assessed following pupillary dilation with
direct ophthalmoscopy until 1989 and afterwards by fundus
photography and graded: nil, simplex, or proliferative
retinopathy. Eighty per cent of the present patients were
evaluated by fundus photo alone.
Albuminuria was measured by radioimmunoassay in all
urine samples until 1992. After 1992 an ELISA method was
used. The correlation between the two methods was r s 0.99.
Serum creatinine concentration was assayed by a kinetic Jaffe
method.
GFR was measured after a single i.v. injection of 3.7 MBq
51
Cr-labeled EDTA by determination of the radioactivity
in venous blood samples taken 180, 200, 220 and 240 min
after the injection w10,11x. Additional blood samples were
obtained after 270 and 300 min, when the GFR dropped
below 20 mluminu1.73 m2. The small underestimation (10%)
of w51CrxEDTA clearance versus clearance of inulin was
corrected for by multiplying EDTA clearance by 1.10 w10x.
Extra renal loss was corrected by subtracting 3.7 mlumin
w10x. We standardized the GFR results for 1.73 m2 body
surface area, using the patients’ surface area at the start of
the studies, for the entire study period. We used the plasma
clearance of w51CrxEDTA for GFR determination because
this method is accurate, precise (coefficient of variation 4%),
and do not require frequent timed urine collections as the
classical renal clearance procedures w10,11x. Furthermore, we
did not use the classical renal plasma clearance of insulin,
because residual urine, which is frequently found in longstanding diabetic patients due to diabetic cystopathy w12x,
may affect the results.
All plasma clearance studies were carried out between
09.00 and 13.30 hours. Patients had their usual breakfast
and morning medication before the investigation, which was
carried out with the patient in supine position. They drank
200 ml tap water per hour during the clearance study.
Arterial blood pressure was measured with a clinical
sphygmomanometer after 10 min of rest. Diastolic blood
pressure was measured at the disappearance of the Korotkoff
sounds (phase V). Arterial hypertension was diagnosed
according to the World Health Organization Criteria,
systolic blood pressure P160 mmHg anduor diastolic blood
pressure P95 mmHg, or if antihypertensive treatment was
being prescribed. Mean arterial blood pressure (MABP)
was calculated as diastolic blood pressure plus one third of
the pulse amplitude.
Body mass index (BMI) was calculated as body
weightuheight2 (kgum2).
Serum lipids, serum total cholesterol and serum highdensity lipoprotein (HDL) cholesterol were measured using
conventional laboratory techniques.
Haemoglobin A1c (HbA1c) was determined by DIAMAT
Analyser, BIO-RAD, California, USA until 1995, and
thereafter by Variant Bio-Rad. Normal range for HbA1c
was 4.1 to 6.1% for both methods.
Follow-up evaluation
Patients were scheduled to visit the outpatient clinic every 3
to 6 months when antidiabetic treatment and antihypertensive therapy were adjusted. During the 7 (1.1–17) years
Statistical analysis
Normally distributed data are expressed as means with
standard deviation (SD) in demographic data or standard
errors of means (SE) in follow-up data. Values for albuminuria were logarithmically transformed before statistical
analysis because of their positively skewed distribution. All
comparisons of normally distributed parameters were done
with a Student’s t-test, using unpaired design between groups
and paired design within groups. A Mann–Whitney’s U-test
was used to evaluate independent samples and a Wilcoxon in
related variables not normally distributed. Fisher’s exact test
was performed in dichotomous variables. Univariate linear
regression and multivariate backwards stepwise linear regression analysis were used to assess the association between the
rate of decline in GFR and putative progression promoters
(GFR at entry, baseline albuminuria, baseline blood pressure, known duration of diabetes, Vv (mesuglom), FF, GuS
(%) and mean values during follow-up of systolic blood
pressure, diastolic blood pressure, albuminuria, total serum
cholesterol, serum HDL cholesterol and HbA1c). All variables significant at P-0.10 in the univariate analyses were
included in the multiple regression analysis.
All calculations were made using SPSS for Windows (SPSS
Inc., Chicago, USA). A P value of -0.05 was considered
significant (two-tailed).
Results
The kidney biopsy revealed diabetic glomerulopathy
in 34 (69%) and normal glomerular structure (n s9) or
glomerulonephritis (n s6) in 31% of the patients.
The clinical features revealed no significant differences in demographic, clinical or laboratory data at
baseline between patients with or without diabetic
nephropathy (Table 1).
Mean age was approximately 55 years and a
male predominance was found in both groups. Most
patients had elevated body mass index and metabolic
control was poor in both groups. HbA1c increased
from 8.3"1.8% to 8.9"1.7% in the DG group
(NS) and from 8.9"2.1% to 9.3"2.2% (NS) in the
NDG-group, even though antidiabetic treatment was
intensified in both groups during follow-up.
Although approximately 1u3 of the patients in both
groups were treated with lipid lowering drugs, mean
serum total cholesterol was elevated in both groups.
There was no significant difference between groups
in number of patients receiving antihypertensive drugs
at baseline or at end of follow-up. Furthermore, the
numbers of patients receiving two or more drugs at the
end of the study in the two groups are not significantly
different. However, all patients in the DG-group (41%)
who did not receive antihypertensive treatment at entry
to the study were prescribed antihypertensive drugs
during follow-up and an increasing number of patients
2340
P. K. Christensen et al.
Table 1. Clinical data in 49 albuminuric type 2 diabetic patients without diabetic retinopathy, with biopsy proven diabetic or non-diabetic
glomerulopathies
Renal structure
Diabetic
glomerulopathy
Number, sex (muf )
27u7
Age (years)
55 (8)
30 (5)
Body mass index (kgum2)
Known duration of diabetes (years)
6 (6)
8.8 (1.5)
Haemoglobin A1c (%) a
6.8 (1.5)
Serum total cholesterol (mmolul) a
1.08 (0.43)
Serum HDL cholesterol (mmolul) a
Antidiabetic treatment (dietuoral hypoglycaemicuinsulin) (%)
At entry
23u57u20
At end
9u29u62
Antihypertensive treatment (niluone drugutwo drugs or more) (%)
At entry
41u26u32
At end
0u15u85
Type of drugs (diureticuACEI or AngII receptor blockeruCa antagonistub-blocker) (%)
At entry
47u27u15u17
At end
88u68u29u24
Retinopathy (nilusimplexuproliferative) (%)
At end
50u47u3
Smoking (%)
53
Prevalence of hypertension (%)
88
P value
Non-diabetic
glomerulopathies
13u2
54 (8)
31 (8)
4 (3)
8.5 (1.5)
6.4 (1.3)
1.04 (0.44)
NS
NS
NS
NS
NS
NS
NS
27u60u13
20u33u47
NS
NS
27u20u53
13u13u73
NS
NS
66u27u27u33
80u73u53u7
NSb
NSb
27u66u7
27
100
NS
NS
NS
Mean (SD).a Calculated from all measurements during follow-up for each patient. b Number of patients treated with ACEI or AngII receptor
blocker.
were at the end of the study treated with two or more
drugs. In patients with non-diabetic glomerulopathies
less profound change in antihypertensive treatment
were seen. Despite a 100% prevalence of hypertension,
13% of patients in this group were not treated
with antihypertensive drug at the end of the study
(Table 1).
The rate of decline in GFR in the DG-group in
patients (n s23) treated with ACE inhibitors or angiotensin II receptors blockers during follow-up was
4.8"0.9 mluminuyear compared to 6.3"1.9 mluminu
year in patients (ns 11) not treated with these drugs
(NS). In the NDG-group the rate of decline in GFR
was 2.2"1.9 mluminuyear vs 3.6"1.0 mluminuyear (NS)
in patients treated with (ns 11) and without (ns 4)
ACE inhibitors or angiotensin II receptors blockers,
respectively.
During the 7 years of follow-up more than 50%
of patients developed retinopathy. Approximately
half of the patients in the DG-group and a third of
the patients in the NDG-group were smokers (NS).
The rate of decline in GFR among smokers and
non-smokers in the DG-group was 5.6"1.1 mluminu
year vs 4.9"1.3 mluminuyear (NS), and in the NDGgroup 0.2"0.4 mluminuyear vs 4.3"1.0 mluminuyear
(P-0.05), respectively.
Data on the course of GFR, serum creatinine,
albuminuria and arterial blood pressure are presented
in Table 2. One patient with previously persistent
albuminuria in the DG-group had albuminuria
-300 mgu24 h at entry, because antihypertensive
treatment was initiated before entry to the study.
All patients had GFR determined at least three times
and the follow-up was more than 1 year in all patients.
Both groups had a significant reduction in GFR
during follow-up (Table 2). The mean difference
(95% confidence interval) in the rate of decline in
GFR between the two groups was 2.1 ( 0.3 to 4.5)
mluminuyear, P s 0.09. Albuminuria decreased significantly in the NDG-group, whereas it remained
unchanged in the DG-group. MABP decreased
from 109"2 to 100"2 mmHg in the DG group
(P-0.001), and from 107"3 to 104"3 mmHg in the
NDG-group (NS).
The individual renal pathology, rate of decline in
GFR, mean albuminuria and mean MABP during
follow-up in patients with diabetic glomerulopathy,
normal glomerular structure and with glomerulonephritis are shown in Tables 3, 4 and 5, respectively.
A detailed description of the renal pathology in all
patients has been presented previously w4x.
An association was found between Vv (mesuglom)
and rate of decline in GFR mainly in the NDGgroup (Fig. 1). The rate of decline in GFR was
associated with FF only in patients with non-diabetic
glomerulopathies (Fig. 2). No significant association
was revealed between the rate of decline in GFR and
the percentage of totally sclerotic glomeruli in any of
the groups (Fig. 3). In the DG-group the rate of
decline of GFR raised with arteriolar hyalinosis
Renal structure and function in diabetic patients
2341
Table 2. Course of GFR, serum creatinine, albuminuria and arterial blood pressure in 49 albuminuric type 2 diabetic patients without
diabetic retinopathy, with biopsy proven diabetic or non-diabetic glomerulopathies
P valuea
Renal structure
Follow-up (months)
Number of GFR measurements
GFR (mluminu1.73 m2)
At entry
At end
P valueb
Rate of decline
Serum creatinine (mmolul)
At entry
At end
P valueb
Albuminuria (gu24 h)
At entry
At end
P valueb
Median during follow-up
Systolic blood pressure (mmHg)
At entry
At end
P valueb
Mean during follow-up
Diastolic blood pressure (mmHg)
At entry
At end
P valueb
Mean during follow-up
Diabetic
glomerulopathy
(n s 34)
Non-diabetic
glomerulopathies
(n s 15)
81 (13–175)
8 (3–20)
84 (35–204)
8 (4–17)
NS
NS
97 (5)
66 (5)
P-0.001
5.3 (0.8)
93 (7)
74 (11)
P-0.01
3.2 (0.9)
NS
NS
86 (51–148)
115 (65–376)
P-0.001
89 (52–130)
108 (70–276)
P-0.01
NS
NS
1322 (169–4731)
1336 (30–9460)
NS
1332 (175–7869)
1491 (341–8739)
716 (24–6253)
P-0.05
1083 (140–5018)
NS
NS
151 (3)
143 (4)
NS
152 (3)
145 (5)
146 (4)
NS
151 (4)
89 (4)
78 (2)
P-0.001
84 (1)
88 (2)
84 (3)
NS
88 (2)
P s 0.09
NS
NS
NS
NS
NS
NS
NS
Median (range) or mean (SE). aSignificance of difference between and bwithin the two groups.
increasing from slight to severe from 3.5"1.7 to
5.9"5.1, NS.
In the DG-group
Univariate analysis revealed significant correlations
between rate of decline in GFR and baseline
systolic blood pressure (r s0.40, P-0.02), and
known duration of diabetes (rs 0.39, P-0.03), but
not with GFR at entry, baseline log10 albuminuria, and
baseline diastolic blood pressure. The same analyses
based on mean values during follow-up of systolic blood
pressure, diastolic blood pressure, log10 albuminuria,
total serum cholesterol, serum HDL cholesterol and
HbA1c, revealed significant correlations between the
rate of decline in GFR and systolic blood pressure
(r s0.34, P s 0.05), and log10 albuminuria (r s0.47,
P-0.01). A multiple regression analysis was performed with known duration of diabetes, Vv (mesu
glom), mean values during follow-up of systolic blood
pressure and log10 albuminuria as independent variables and rate of decline in GFR as dependent
variable. The mean log10 albuminuria during follow
was the only variable associated with the rate of
decline in GFR (rs 0.40, P-0.02).
In the NDG-group
The same variables as mentioned above were introduced in the univariate analyses, and correlations
between the rate of decline in GFR and GFR at entry
(r s 0.64, P-0.02), baseline log10 albuminuria
(r s0.58, P-0.05), Vv (mesuglom) (rs 0.74, P-0.01),
mean values during follow-up of systolic blood
pressure (rs 0.63, P-0.01) and log10 albuminuria
(r s0.69, P-0.02) were found. A multiple regression analysis was then performed with GFR at entry,
Vv (mesuglom), FF and mean values during follow-up
of systolic blood pressure and log10 albuminuria as
independent variables, and rate of decline in GFR
as dependent variable. The mean log10 albumniuria
during follow (rs 0.61, P-0.01) and GFR at entry
(r s 0.53, P-0.02) were the only variables associated
with the rate of decline in GFR.
Albuminuria
Univariate analyses were furthermore performed
including; Vv (mesuglom), FF, SuG, and mean values
during follow-up of MABP, and HbA1c as independent
variables and mean log10 albuminuria as dependent
variable. The analyses were performed in both groups,
2342
P. K. Christensen et al.
Table 3. Renal pathology in 34 albuminuric type 2 diabetic patients without diabetic retinopathy and with diabetic glomerulopathy
No.
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Light and electron microscopy
Diabetic
glomerulosclerosis
Grading
of severity
of DGS
Vv
(mesuglom)
FF
SuG
(%)
Arteriolar
hyalinosis
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
Diffuse
1
1
1
3
2
1
1
1
2
1
2
2
3
1
1
1
1
1
1
3
1
1
1
1
2
1
1
1
2
2
1
1
2
1
0.24
0.31
0.38
0.39
0.42
0.27
0.25
0.25
0.41
0.28
0.37
0.38
0.38
0.31
0.30
0.27
0.32
0.30
0.30
0.36
0.30
0.34
0.37
0.29
0.36
0.27
0.38
0.30
0.43
0.36
0.24
0.30
0.53
0.33
0.02
0.01
0.25
0.14
0.04
0.03
0.58
0.11
0.02
0.10
0.04
0.04
0.03
0.55
0.01
0.09
0.09
0.05
0.06
0.09
0.04
0.11
0.01
0.06
0.26
0
0.14
0.07
0.08
0.09
0.08
0.01
0.38
0.08
10
0
25
15
29
11
20
6
9
0
8
25
40
80
0
0
0
0
44
19
4
13
5
6
42
0
23
50
30
17
20
9
18
13
3
1
2
3
2
1
1
1
3
2
2
2
3
3
1
2
3
3
3
3
3
2
3
1
3
1
2
2
–
1
1
2
3
1
Rate of
decline
in GFR
(mluminuyear)
Median
UAER
during
follow-up
(mgu24 h)
Mean
MABP
during
follow-up
(mmHg)
4.7
2.5
0.2
6.0
2.8
2.9
1.4
1.8
5.6
3.1
2.8
15.6
20.0
2.6
4.4
2.0
9.4
3.2
4.7
10.3
4.2
8.6
0.2
4.6
2.4
1.8
3.4
5.6
20.3
5.8
3.7
5.2
2.6
6.2
245
1380
1836
5525
1354
373
1316
544
444
16 920
546
2010
7869
1210
482
392
837
1081
175
3567
295
3086
1296
464
1478
1111
1897
1416
2777
1389
2412
4578
1067
1348
101
112
124
117
99
103
106
99
99
109
108
109
122
107
90
107
106
104
101
106
103
112
96
104
104
98
126
105
104
106
113
108
105
118
DGS, diabetic glomerulopathy; Vv (mesuglom), mesangial volume as % of the total glomerular volume; FF, fractional area of focal cortical
interstitial fibrosis; SuG, percentage of sclerosed glomeruli of the total number of glomeruli in each biopsy; slight s 1, medium s 2, severe s 3;
ND, no data; UAER, urinary excretion rate.
Table 4. Renal pathology in nine albuminuric type 2 diabetic patients without diabetic retinopathy and with normal glomerular structure
No.
35
36
37
38
39
40
41
42
43
Immune depositsa
Light and electron microscopy
Vv
(mesuglom)
FF
SuG
(%)
Arteriolar
hyalinosis
Immunoglobulins
Complement
fractions
TI
0.23
0.27
0.23
0.26
0.30
0.19
TI
0.17
0.08
0.08
0.04
0.01
0
0.08
0.06
0.07
0.20
33
17
0
0
0
8
33
50
33
1
1
1
0
2
1
2
3
2
0
0
0
0
0
0
IgA
IgM
ND
0
0
0
0
0
0
0
C1, C3, C4
ND
Rate of
decline
in GFR
(mluminuyear)
4.5
1.3
4.1
0.2
4.1
7.3
0.5
6.0
0.8
Median
UAER
during
follow-up
(mgu24 h)
Mean
MABP
during
follow-up
(mmHg)
4005
1890
1083
551
185
1959
140
1217
808
114
112
104
99
99
109
114
126
107
Vv (mesuglom), mesangial volume as percentage of the total glomerular volume; FF, fractional area of focal cortical interstitial fibrosis; SuG,
percentage of sclerosed glomeruli of the total number of glomeruli in each biopsy; slight s 1, medium s 2, severe s 3; ND, no data;
TI, technical impossible; UAER, urinary albumin excretion rate. aAll deposits of immunoglobulins and complement fractions were mild
or questionable, frequently only present in a few glomeruli and without characteristic pattern.
No.
Light and electron microscopy
Diabetic
glomerulosclerosis
Vv
(mesuglom)
FF
Immunofluorescence microscopy
SuG
(%)
Arteriolar
hyalinosis
44
Absent
0.22
0.01
14
2
45
Absent
0.24
0.05
20
3
46
Absent
TI
0.01
8
3
47
Diffuse
0.31
0.02
TI
0
48
Nodular
0.37
0.24
56
3
49
Diffuse
0.23
0
0
1
Non-diabetic
glomerular
lesions
Mild mesangial
prolif. GN (IgA?)
GN
sequelae
Moderate
mesangial
prolif. GN
Probably IgA
nephropathy
IgA
nephropathy
IgA
nephropathy
Rate of
decline in
GFR
(mluminuyear)
Median
UAER
during
follow-up
(mgu24 h)
Mean
MABP
during
follow-up
(mmHg)
Immune deposits
Localization
Immunoglobulins
Complement
fractions
Mesangium
Capillary
IgA, IgM
C3
q
q
0.6
1359
102
IgM
0
q
q
7.7
5018
111
0
C3
q
q
0.2
168
117
IgA, IgM
0
q
q
1.8
788
102
IgA, IgM
0
q
q
10.2
4798
119
IgA, IgM
C3, C4
q
q
1.3
624
102
Renal structure and function in diabetic patients
Table 5. Renal pathology in 6 albuminuric type 2 diabetic patients without diabetic retinopathy and with glomerulonephritis
DGS, diabetic glomerulosclerosis; Vv (mesuglom), mesangial volume as % of the total glomerular volume; FF, fractional area of focal cortical interstitial fibrosis; SuG, percentage of sclerosed
glomeruli of the total number of glomeruli in each biopsy; slight s 1, medium s 2, severe s 3; ND, no data; TI, technical impossible; UAER, urinary albumin excretion rate.
2343
2344
Fig. 1. Mesangial volume expressed as fraction of total glomerular
volume in albuminuric type 2 diabetes patients without retinopathy
in relation to rate of decline in glomerular filtration rate. m Patients
with diabetic glomerulopathy (r s 0.31, P s 0.07). h Patients with
glomerulonephritis or j normal glomerular structure (r s 0.74,
P-0.01).
and revealed that Vv (mesuglom) was associated to
mean log10 albuminuria during follow-up (DG group;
rs 0.38, P-0.03 and NDG group; r s0.51, P s0.09).
Furthermore log10 albuminuria was associated with
average MABP during follow-up (rs 0.56, P-0.01) in
the DG group, but not in the NDG group. Metabolic
control was not related to mean log10 albuminuria
during follow-up in any of the groups.
Discussion
The present long-term observational study of albuminuric patients with type 2 diabetes without diabetic
retinopathy showed a large variation in rate of decline
in GFR and a tendency to a faster rate of decline in
kidney function in patients with biopsy proven diabetic
glomerulopathy (DG-group) compared to patients
with normal glomerular structure (ns 9) or glomerulonephritis (n s 6). Albuminuria was reduced in the
NDG-group despite unchanged blood pressure during
follow-up, whereas albuminuria remained unchanged
during follow-up despite intensified antihypertensive
treatment and blood pressure reduction during
follow-up in the DG-group. The differences in
blood pressure and antihypertensive treatment might
be responsible for the lack of significant differences in
rate of decline in GFR and albuminuria at the end
P. K. Christensen et al.
Fig. 2. Fractional area of focal interstitial fibrosis and tubular
atrophy of cortical area in albuminuric type 2 diabetes patients
without retinopathy in relation to rate of decline in glomerular
filtration rate. m Patients with diabetic glomerulopathy (r s 0.24,
NS). h Patients with glomerulonephritis or j normal glomerular
structure (r s 0.48, P s 0.07).
of the study. However, a close correlation between
albuminuria and rate of decline in GFR was demonstrated in both groups. Several other studies have
shown that albuminuria and blood pressure are
independent risk factors for the progression of renal
diseases w13–15x, in agreement with our results.
Vv (mesuglom) was the main structural predictor of
the rate of decline in GFR and albuminuria during
follow-up. The histological classification of the renal
lesions was performed according to the World Health
Organization standard. All biopsies were reviewed by
three masked and experienced nephropathologists.
Since only a part of our biopsies included tissue for
EM, we used a morphometric point count technique
for estimation of Vv (mesuglom) on light microscopic
specimens. Furthermore, the conventional method of
measuring fractional volume of cortical fibrosis in
biopsies with rather few and small focal fibrotic areas
is very insensitive since the fibrosis will largely
‘disappear’ in the many measured fields outside the
areas with fibrosis. Therefore, we also selected another
objective to evaluate fibrosis.
It must be emphasized, that the sole indication
for kidney biopsy in our study was the presence of
albuminuria and lack of diabetic retinopathy. Selection bias in the present study may be possible, since
15 patients with albuminuria -1 gu24 h were not
included. However, these patients were not different
Renal structure and function in diabetic patients
Fig. 3. Percentage of sclerosed glomeruli of the total number of
glomeruli in each biopsy from albuminuric type 2 diabetic
patients without retinopathy in relation to glomerular filtration
rate. m Patients with diabetic glomerulopathy (r s 0.21, NS).
h Patients with glomerulonephritis or j normal glomerular
structure (r s 0.42, NS).
to the included patients with respect to demographic,
clinical or laboratory data.
Recently, six studies evaluating clinical and histological correlations of the course of renal function have
been reported in albuminuric patients with type 2
diabetes w3,5–9x. However, the results from the studies
are difficult to compare because of differences in the
methods used for estimations of renal structure and
renal function.
One study has estimated the renal lesions by
combining the basement membrane thickness and Vv
(mesuglom) w5x, while others have combined glomerular
and vascular and interstitial changes w7x. Both these
studies showed a relationship between the course of
kidney function and renal structure. Two studies have
divided patients in accordance to histology diagnosis
w6,7x. One long-term (7.7 years) study revealed a faster
rate of decline in GFR in albuminuric type 2 diabetic
patients with diabetic glomerulopathy compared to
patients without glomerulopathy w6x, whereas a shortterm study (1.8 years) did not show such a relationship
w7x. Finally, three studies have used individual measurements such as glomerular basement membrane
width, Vv (mesuglom), mean glomerular volume,
interstitial fibrosis, capillary volume, percent of sclerosed glomeruli and cortical interstitium fractional
volumes w3,8,9x. Their results suggested that cortical
2345
interstitium fractional volumes, glomerular basement
membrane width, Vv (mesuglom) and interstitial
fibrosis might be related to the course of kidney
function, but results are conflicting. The abovementioned results should be interpreted with caution, since they are based on mixed populations of
patients with micro- or macroalbuminuria w3,9x, type 1
or type 2 diabetes w5,8x and with or without retinopathy w3,5–9x. Only one study includes more than
40 albuminuric type 2 diabetic patients, however the
follow-up was less than 2 years w7x. Even though there
are differences and limitations, the results from previous and the present study show that kidney biopsy
has a prognostic role in albuminuric type 2 diabetic
patients. Furthermore, exact knowledge of the underlying cause of albuminuria may play an important
role in offering the correct treatment, as demonstrated in patients with type 2 diabetes suffering
from non-diabetic glomerulopathies w16–18x or diabetic
glomerulopathy w3x.
The most characteristic and clinically important
glomerular lesion in diabetes mellitus is mesangial
expansion. In agreement with previous findings, we
found a tendency of a relationship between Vv
(mesuglom) and rate of decline in GFR. This relationship probably resulted from the expanding mesangium
compromising the structure of glomerular capillaries
and reducing the ultrafiltration coefficient filtration
(defined as the product of effective hydraulic permeability and total glomerular capillary surface area
of the kidney), as demonstrated in a pathophysiological
study of albuminuric Pima Indians with diabetes w19x.
A correlation between FF and rate of decline in GFR
was found in the patients without diabetic glomerulopathy, indicating that the interstitial lesions are
important determinants of the progression in kidney
disease, as suggested by Mauer w20x.
Large glomeruli have been found in elderly albuminuric type 2 diabetic patients, indicating that they are
capable of producing glomerular growth w5x. There are
good reasons to believe that this represents a compensatory hypertrophy, which is induced in order to
prevent loss of filtration surface. This may explain the
lack of association between the rate of decline in GFR
and the percentage of totally sclerotic glomeruli found
in our study.
Several subjects with diabetic glomerulopathy had
marked mesangial lesions anduor marked interstitial
lesions anduor severe glomerulosclerosis but only
modest reduction in GFR; but it was not possible to
find a pattern within this group. Other studies have
calculated a global score of tissue injury, but even
when renal tissue lesions are combined in a global
score system the underlying glomerular lesions have
less predictive value in patients with heavy albuminuria
w7x. These findings are in agreement with our study,
where patients with heavy albuminuria irrespectively
of the underlying glomerular lesions had a rather fast
rate of decline in GFR.
A small subgroup of patients without diabetic
glomerulopathy had moderate or severe arteriolar
2346
hyalinosis and a great relative number of sclerosed
glomeruli. This is not uncommon in kidneys from nondiabetic persons with normal renal function and no
albuminuria in the same age group as our patients w21x.
Furthermore, due to the restricted number of glomeruli in a biopsy, the relative number of sclerosed glomeruli present in a biopsy is subjected to considerable
sampling error. The rate in decline of GFR compared
with the relative number of sclerosed glomeruli in
the biopsy in our group of patients without diabetic
glomerulopathy did not reveal a significant pattern.
Albuminuria and deterioration of renal function in
some of our patients with normal renal structure may
reflect one of the following causes: minimal-change
nephropathy, focal segmental glomerulosclerosis
(undetected), silent diabetic glomerulosclerosis (electron microscopic glomerulopathy) and finally, a
hypothetic entity with normal renal structure but
increased glomerular permeability to macromolecules
(sizeucharge defects). Furthermore arteriolar structural abnormalities may lead to impaired vascular
responses to changes in systemic blood pressure and
thereby contribute to increased leakiness of glomerular
filter w22,23x.
Urinary albumin excretion is the net result of
interactions of factors within glomeruli as well as
extra-glomerular renal and systemic conditions w24x,
which makes it difficult to establish the pathogenesis
of albuminuria in individual subjects.
The marked heterogeneity of severity of glomerular
disease combined with marked differences in severity
of albuminuria and blood pressure elevation might
explain the difference in rate of decline in GFR seen in
patients with non-diabetic kidney diseases. A rate of
decline in GFR close to the age dependent decline in
kidney function seen in normal subjects was revealed in
patients who had minimal structural lesion and low
levels of albuminuria combined with relatively low
blood pressure. These findings are in agreement with
other studies evaluating renal structure and function as
mentioned above. However, the data on the structural
and functional relationship in patients with both
diabetic and non-diabetic kidney disease are limited
and such a subgroup needs to be studied in the future.
The impact of metabolic control on the progression
of kidney disease in type 2 diabetic patients is debated.
In agreement with our findings several other studies
have failed to demonstrate a significant correlation
between glycaemic control and rate of decline in
GFR in albuminuric type 2 diabetic patients w19,25x.
In contrast, studies in type 1 diabetic patients have
shown that hyperglycaemia is a progression promoter
of diabetic nephropathy w26,27x.
We found no difference in the rate of decline in
GFR between patients treated with or without ACE
inhibitors or angiotensin II receptors blockers, but a
variety of antihypertensive drugs were used during
follow-up. The WHO criteria of hypertension from
1978 were used to describe our patients at baseline,
several new criteria have since then reduced the arterial
blood pressure limit for hypertension. Consequently
P. K. Christensen et al.
antihypertensive treatment was initiated and increased
at different time points. It is therefore possible that
the differences in antihypertensive treatment may
have induced a bias in the structural and functional
relationships despite the above-mentioned findings.
Two patients in the NDG-group with isolated
systolic hypertension did not receive antihypertensive
treatment during follow-up, since they died before
1996 and data justifying such therapy was lacking
until 1996 w28x.
In agreement with our findings, the effect of
smoking on the progression in kidney disease is not
clear; while some studies w29,30x do not support smoking as a putative progression promoter others have
shown that smoking may increase the progression of
nephropathy in diabetic patients w31x.
Our study has several limitations including a relatively small number of patients, selection of patients
without retinopathy may result in less glomerulopathy
compared to patients with retinopathy w5x, lack of
tissue for electron microscopy material made our
histological evaluation less precise, the biopsies only
provides a ‘snap-shot’ and sequential biopsies would
probably provide more complete information on the
evolution profile of the underlying diseases, and finally
the design and power of the study make it impossible
to evaluate the effect of different antihypertensive
regimens on rate of decline in GFR.
In conclusion, our study revealed a tendency to
a faster rate of decline in GFR in the DG-group
compared to the much smaller NDG-group, characterized by marked heterogeneity of the underlying
kidney lesions and rate of GFR loss. A large mesangial volume fraction was associated with increased
albuminuria and loss in GFR. Albuminuria acted
as progression promoter in both groups.
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Received for publication: 6.3.01
Accepted in revised form: 19.7.01