1 Steno Diabetes Center, Gentofte and 2 Department of Pathology, Herlev Hospital, Herlev, Denmark
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Abstract |
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Methods. Mesangial volume of total glomerular volume (Vv (mes/glom)), fractional area of focal interstitial fibrosis and tubular atrophy of cortical area (FF) and percentage of sclerosed glomeruli (S/G) 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 320)) in each patient. Patients were followed for 7.0 (1.117) years. Albuminuria and blood pressure were measured every 36 months.
Results. Biopsies revealed diabetic glomerulopathy (DG-group) in 69% of the patients (27 male/7 female) and normal glomerular structure (n=9) or glomerulonephritis (n=6) were found in 31% (13 male/2 female) (NDG-group). In the DG-group GFR decreased from 97±5 to 66±5 ml/min/1.73 m2 (mean±SE) (P<0.001), with a rate of decline in GFR of 5.3±0.8 ml/min/year and in the NDG-group from 93±7 to 74±11 ml/min/1.73 m2 (P<0.01), with a rate of decline in GFR of 3.2±0.9 ml/min/year, P=0.09 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 NDG-group. An association between Vv (mes/glom) and rate of decline in GFR was revealed mainly in the NDG-group (DG-group; r=0.31, P=0.07 and NDG-group; r=0.74, P<0.01). Furthermore, the rate of decline in GFR seemed to be associated with FF in the NDG group (r=0.48, P=0.07). Percentage of S/G was not associated with the rate of decline in GFR. Vv (mes/glom) was associated with mean albuminuria during follow-up in the DG group; r=0.38, P<0.03 (NDG group; r=0.51, P=0.09). Albuminuria was an independent predictor of the rate of decline in GFR in both groups (DG-group; r=0.40, P<0.05 and NDG-group; r=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
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Introduction |
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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 [4].
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Subjects and methods |
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All Caucasian (n=347) patients with type 2 diabetes and persistent albuminuria (>300 mg/24 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 g/24 h. Consequently 15 patients without retinopathy with albuminuria <1 g/24 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=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 one patient it was not possible to estimate the rate of decline in GFR, because fewer than three GFR measurements were performed.
All (n=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 exclude/minimize 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 [4]. Diffuse diabetic glomerulopathy, hyaline arteriolosclerosis, tubular atrophy, and interstitial fibrosis were semiquantitatively estimated. The final reading (0, +1, +2, +3) 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 (mes/glom)) was measured at light microscopy by point counting using a computer-assisted stereological system (GRID, Interactivision, DK). Magnification on the video screen was x1650. The grid had 5x6 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 µ section from each biopsy. One field from each of 610 glomeruli was measured (corresponding to 180300 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 µm sections were cut at 24°C on a Leitz Histocryotome (Wetzlar, Germany). Direct immunofluorescent staining technique was applied, using FITC-conjugated rabbit or goat antisera specifically reactive to human IgG, IgM, IgA as well as complement C1q, C3 and C4.
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=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 51Cr-labeled EDTA by determination of the radioactivity in venous blood samples taken 180, 200, 220 and 240 min after the injection [10,11]. Additional blood samples were obtained after 270 and 300 min, when the GFR dropped below 20 ml/min/1.73 m2. The small underestimation (10%) of [51Cr]EDTA clearance versus clearance of inulin was corrected for by multiplying EDTA clearance by 1.10 [10]. Extra renal loss was corrected by subtracting 3.7 ml/min [10]. 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 [51Cr]EDTA 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 [10,11]. Furthermore, we did not use the classical renal plasma clearance of insulin, because residual urine, which is frequently found in long-standing diabetic patients due to diabetic cystopathy [12], 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 160 mmHg and/or diastolic blood pressure
95 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 weight/height2 (kg/m2).
Serum lipids, serum total cholesterol and serum high-density 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 (1.117) years follow-up GFR was measured 8 times (320), albuminuria 29 (475), arterial blood pressure 30 (590), HbA1c 20 (420), total serum cholesterol 11 (130) and serum HDL cholesterol 8 (118) times (medians (ranges)).
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 MannWhitney'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 (mes/glom), FF, G/S (%) 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).
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Results |
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The clinical features revealed no significant differences in demographic, clinical or laboratory data at baseline between patients with or without diabetic nephropathy (Table 1).
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Although approximately 1/3 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 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=23) treated with ACE inhibitors or angiotensin II receptors blockers during follow-up was 4.8±0.9 ml/min/year compared to 6.3±1.9 ml/min/year in patients (n=11) not treated with these drugs (NS). In the NDG-group the rate of decline in GFR was 2.2±1.9 ml/min/year vs 3.6±1.0 ml/min/year (NS) in patients treated with (n=11) and without (n=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 ml/min/year vs 4.9±1.3 ml/min/year (NS), and in the NDG-group 0.2±0.4 ml/min/year vs 4.3±1.0 ml/min/year (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 mg/24 h at entry, because antihypertensive treatment was initiated before entry to the study.
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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 [4].
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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=-0.64, P<0.02), baseline log10 albuminuria (r=0.58, P<0.05), Vv (mes/glom) (r=0.74, P<0.01), mean values during follow-up of systolic blood pressure (r=0.63, P<0.01) and log10 albuminuria (r=0.69, P<0.02) were found. A multiple regression analysis was then performed with GFR at entry, Vv (mes/glom), 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 (r=0.61, P<0.01) and GFR at entry (r=-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 (mes/glom), FF, S/G, 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, and revealed that Vv (mes/glom) was associated to mean log10 albuminuria during follow-up (DG group; r=0.38, P<0.03 and NDG group; r=0.51, P=0.09). Furthermore log10 albuminuria was associated with average MABP during follow-up (r=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.
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Discussion |
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Vv (mes/glom) 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 (mes/glom) 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 g/24 h were not included. However, these patients were not different 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 [3,59]. 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 (mes/glom) [5], while others have combined glomerular and vascular and interstitial changes [7]. 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 [6,7]. 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 [6], whereas a short-term study (1.8 years) did not show such a relationship [7]. Finally, three studies have used individual measurements such as glomerular basement membrane width, Vv (mes/glom), mean glomerular volume, interstitial fibrosis, capillary volume, percent of sclerosed glomeruli and cortical interstitium fractional volumes [3,8,9]. Their results suggested that cortical interstitium fractional volumes, glomerular basement membrane width, Vv (mes/glom) and interstitial fibrosis might be related to the course of kidney function, but results are conflicting. The above-mentioned results should be interpreted with caution, since they are based on mixed populations of patients with micro- or macroalbuminuria [3,9], type 1 or type 2 diabetes [5,8] and with or without retinopathy [3,59]. Only one study includes more than 40 albuminuric type 2 diabetic patients, however the follow-up was less than 2 years [7]. 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 [1618] or diabetic glomerulopathy [3].
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 (mes/glom) 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 [19]. 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 [20].
Large glomeruli have been found in elderly albuminuric type 2 diabetic patients, indicating that they are capable of producing glomerular growth [5]. 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 and/or marked interstitial lesions and/or 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 [7]. 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 hyalinosis and a great relative number of sclerosed glomeruli. This is not uncommon in kidneys from non-diabetic persons with normal renal function and no albuminuria in the same age group as our patients [21]. 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 (size/charge 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 [22,23].
Urinary albumin excretion is the net result of interactions of factors within glomeruli as well as extra-glomerular renal and systemic conditions [24], 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 [19,25]. In contrast, studies in type 1 diabetic patients have shown that hyperglycaemia is a progression promoter of diabetic nephropathy [26,27].
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 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 [28].
In agreement with our findings, the effect of smoking on the progression in kidney disease is not clear; while some studies [29,30] do not support smoking as a putative progression promoter others have shown that smoking may increase the progression of nephropathy in diabetic patients [31].
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 [5], 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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Notes |
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References |
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