Follow-up study of glomerular dimensions and cortical interstitium in microalbuminuric type 1 diabetic patients with or without antihypertensive treatment

Ruth Østerby1,, Hans-Jacob Bangstad2 and Susanne Rudberg3

1 Electron Microscopy Laboratory, Institute of Experimental Clinical Research and Institute of Pathology, Aarhus University, Denmark, 2 Pediatric Department, Ullevål University Hospital, Oslo, Norway, and 3 Department of Woman and Child Health, Pediatric Unit, Karolinska Institute, Stockholm, Sweden



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. A decrease in urinary albumin excretion is regularly seen with antihypertensive treatment in patients with diabetic nephropathy. Our study concerns structural data obtained by light microscopy in baseline and follow-up biopsies in antihypertensive treated patients and in a reference group.

Methods. Microalbuminuric type 1 diabetic patients with diabetes duration of 6–16 years were studied. Two groups, allocated to treatment with either angiotensin-converting enzyme-inhibitor (group 1, n=6) or ß-blocker (group 2, n=6) after the baseline biopsy, were studied in parallel, whereas the reference group (group 3, n=9), without antihypertensive treatment, was part of a previously completed study. The renal plastic-embedded biopsies were serially sectioned (1 µm), the sections being used for determining glomerular volume, vascular pole area, and interstitial space expressed as fraction of tubular cortex.

Results. A significant increase in glomerular volume (P=0.04) was seen in group 3 only. Vascular pole area (VPA) and VPA relative to calculated glomerular surface did not show significant changes in any of the groups, only a tendency to increase in VPA in group 3 (P=0.051). The increase in VPA correlated with systolic blood pressure during the study period (r=0.49, P=0.03). Glomerular volume did not correlate with HbA1C, current diabetic glomerulopathy, or ensuing worsening of glomerulopathy. In group 3 every case showed an increase in interstitium (P=0.0009), group 2 showed a decrease (P=0.03), and group 1 showed no change. Increase in interstitial fractional volume correlated with diastolic blood pressure during the study (r=0.54, P=0.01).

Conclusions. In early microalbuminuria, type 1 diabetic patients show glomerular growth, probably compensatory to the developing glomerulopathy. The increase in interstitial volume fraction, demonstrable in early nephropathy, is further augmented over a few years, but is arrested by antihypertensive treatment.

Keywords: antihypertensive treatment; diabetic glomerulopathy; glomerular size; glomerular vascular pole; microalbuminuria; renal interstitium



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Studies of structural data from sequential biopsies of native kidneys in diabetic patients are very few [17]. Therefore, the natural history of the development of renal lesions is for the large part illustrated from cross-sectional studies of clinically defined groups of patients [810]. A more detailed knowledge of patterns of development could be provided in repeat biopsies in individual patients.

Antihypertensive treatment has been widely used for a number of years, and its beneficial effect on the progression of nephropathy is well established [11]. Reports on the possible effect on renal structures are beginning to appear [6,7,12]. From studies in experimental animals it was concluded that one effect of angiotensin-converting enzyme (ACE) inhibitors is a reduction of efferent arteriolar resistance [13,14]. Whether this is the case in patients cannot be deduced from animal data. At any rate, it seems of interest to determine glomerular dimensions before and after such treatment. The present series of follow-up biopsies from type 1 diabetic patients with microalbuminuria obtained before and after a period of antihypertensive treatment with either ACE inhibitor or ß-blocker enabled us to address this question.

Follow-up studies of glomerular volume are also of interest with the suggestion of a causal effect of glomerular hypertrophy on the ensuing development of glomerulopathy in a variety of renal diseases [1518]. In the present study, comparisons were made between glomerular volume vs progression in ultrastructural parameters of the diabetic glomerulopathy. Detailed results on the latter are presented in another paper [6].

Biopsy results in type 2 diabetic patients indicate that ACE inhibitors may prevent the progression of interstitial expansion [12], and analogous effects have been demonstrated in several experimental settings [19,20]. These observations are particularly noteworthy since great emphasis has been put on the role of changes in the interstitium in the development towards renal functional impairment [4,21]. The present study includes quantitation of interstitial volume fraction in the follow-up biopsies.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
The patients were recruited from three departments of paediatrics in Stockholm. Inclusion criteria were age >=15 years with >5 years of type 1 diabetes, and microalbuminuria defined as albumin excretion rate (AER)>15 µg/min in at least two of three consecutive overnight urine samples during the preceding year. The duration of microalbuminuria was short-term in all cases, less than 2 years before the start of the study. Eighteen microalbuminuric patients entered the study and had a baseline renal biopsy taken. The patients were then consecutively allocated to treatment with either the ACE-inhibitor enalapril (20 mg/day, group 1) or the ß-blocker metoprolol (100 mg/day, group 2) according to a randomization list. Five patients dropped out during the treatment period due to pregnancy (two patients), social reasons (two patients) and severe hypertension that required multiple antihypertensive drugs (one patient). In one case in group 1 there was insufficient material for the present measurements, so that the final material of follow-up biopsies was six patients in each of groups 1 and 2.

Control group
Since a non-treated control group was not accepted by the ethics committee we used as a reference group (group 3) a previously obtained biopsy series comprising type 1 diabetic patients with the same age, duration of diabetes, and levels of microalbuminuria as in the present series. These patients were part of a previously conducted controlled study of the effect of intensified insulin treatment [3]. The present subset received only conventional diabetes treatment with multiple daily insulin injections during the follow-up period. The reference group was therefore not part of the design of the study of antihypertensive treatment, and due to the non-contemporary study period it is not the optimal, but the best available, reference group.

Clinical data
The clinical data for the three groups at the time of the baseline biopsy are presented in Table 1Go in which pooled data for groups 1 and 2 are given. Only those patients are included in whom both baseline and follow-up biopsies were obtained. No significant differences were found between the groups in any of the clinical variables. For comparison between the groups at outset, the ultrastructural estimates of glomerulopathy are also presented (Table 2Go), as previously published [22,23]. Also in terms of glomerulopathy the groups were comparable, with no significant differences. Likewise, for the patients subsequently allocated to treatment with ACE inhibitor (group 1), or with ß-blocker (group 2), no significant differences were seen at the outset, except for age: in group 1 on average 18 years vs 20 years in group 2, (P=0.03).


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Table 1. Clinical data at baseline in two groups before antihypertensive treatment (groups 1 and 2) and the reference group (group 3)

 

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Table 2. Baseline data on glomerulopathy

 
Prior to the renal biopsy at baseline and at follow-up, GFR was determined with continuous inulin clearance in all three groups. During the treatment period AER (immunoturbidimetric method), blood pressure (Dinamap), and HbA1C (HPLC) were determined every third month in groups 1 and 2 [6]. In group 3 AER and HbA1C were measured every second month using the same methods as in groups 1 and 2. For HbA1C the reference levels were 4.0–6.0 and 4.3–6.1 at the two clinics respectively. Blood pressure was also measured every second month, using conventional sphygomanometry. The second biopsy was obtained after a treatment period of 40 months (range 36–48 months) in groups 1 and 2 and after 30 months (range 26–34 months) in group 3. Clinical data concerning the follow-up period are presented in Table 3Go.


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Table 3. Clinical data during study period

 

Biopsies
Percutaneous needle biopsies were obtained and immediately fixed in buffered 2% glutaraldehyde. The biopsy material was mailed in the fixative to the laboratory in Aarhus where the processing for light and electron microscopy was performed. For the antihypertensive-treated groups the embedding medium was epon 112 (TAAB Laboratories, Berkshire, England), and for the reference group vestopal (Serva Feinbiochemica, Heidelberg, Germany), in all groups the same embedding material was used in baseline and follow-up biopsies.

Consent
Informed consent was obtained in each case and the studies were approved by the local committees of ethics.

Quantitations by light microscopy
The plastic embedded blocks were serially sectioned with 1-µm-thick sections which were all picked up on slides and stained with toluidine blue. Every tenth section, i.e. an interval of 10 µm, was used for glomerular volume and vascular pole area measurements. The real obtained distance between these levels was measured as previously described [24]. In brief, a terrace was cut about 300–400 µm deeper to the plane of sectioning when starting cutting the block, and the distance between section plane and the terrace was re-measured using the steps of the ultramicrotome after having cut through the tissue.

Glomerular volume (VG)
Only new appearing glomeruli were used for determination of glomerular volume, i.e. sampling independent of size. Glomerular volume was estimated with Cavalieri's method [25]: measurement of profile areas 10 µm apart, defined as the convex circumscribed polygon [26]. The first profile used was at a multiple of 10 µm from the baseline section in the block, i.e. at a random level within 0–10 µm from the top of that tuft. The measurements were performed with a light microscope with a drawing tube attached (Olympus BH-2) at 290x magnification, using a square grid with 34 µm distance between points. The total number of glomeruli obtained per biopsy was on average 9–12 in the groups (range 6–26). The volume of individual glomeruli is: t*{Sigma}A, where A is the area of glomerular profiles which equals points hitting the glomerular profile, * the area corresponding to 1 point, 342 µm2, and t=distance between levels, ~10 µm.

Vascular pole area (VPA)
An approximate estimate was obtained by measuring chords through the vascular pole as previously described in detail [24] (Figure 1Go). The size of VPA was calculated as the sum of chords factored by the distance between sectioning levels.



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Fig. 1. Glomerular profile cut at a level traversing the vascular pole. The chord measured at this level for the determination of vascular pole area is indicated.

 
In individual corpuscles where both glomerular volume and VPA were obtainable, the size of VPA was calculated relative to glomerular surface (GS): glomerular volume provides an estimate of glomerular diameter, d=[V(G)*6/{pi}]1/3; GS={pi}*d2. VPA/GS, being a dimensionless ratio of two areas, is expressed as percentage. In one case (group 2) these data were not obtainable due to too low a number of vascular poles cut exhaustively in the biopsy.

Interstitial volume fraction
For this estimate five to eight 1-µm sections were measured by point counting in each biopsy (Figure 2Go). The level predetermined for measurement was that 10 µm deeper than the first level in the block. The whole block face without trimming was used for the point counting. The visual fields were projected from the microscope (Zeiss Axioskop) to a computer screen at a magnification of 725x together with a 9 : 1 grid. The distance between fine points in the grid was 50 µm. Coarse points were counted for hits on any cortical tissue and fine points counted separately hitting each of the components: total interstitial space, glomeruli including Bowman's capsule, large blood vessels, and arterioles. Interstitium was expressed as volume fraction of ‘tubular cortex’, i.e. total cortex minus glomeruli and large blood vessels. The tubular basement membranes were included in interstitial space for the most consistent definition. The average total cortical area measured per biopsy was 4.3x106 µm2 (range 1.6–7.9x106 µm2).



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Fig. 2. 1-µm-thick epon section stained with toluidine blue used for the determination of interstitial volume fraction. The compartments identified are total cortex, interstitial space, capillaries within interstitial space, glomeruli including Bowman's capsule, large blood vessels, and arterioles. The compartment used as reference volume is tubular cortex=total cortex minus glomeruli minus large blood vessels.

 

Statistical methods
Comparisons between data in baseline and follow-up biopsies were tested with a paired Student's t-test. Differences among the three groups were evaluated by the non-parametric Kruskal–Wallis test. When this test showed significant differences, results in groups were compared with the aid of the Mann–Whitney two-sample test. Correlations were studied by simple least-squares regression.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Clinical course
Clinical data obtained during the study period are presented in Table 3Go. Since AER showed a gradual decrease during the initial part of the study period in groups 1 and 2, data are given for the time of follow-up. No significant change in systolic or diastolic pressure was detectable during the antihypertensive treatment in this series of initially normotensive subjects, although there was a tendency to lower diastolic pressure. In groups 1 and 2, AER decreased markedly, and all patients were normoalbuminuric at termination, whereas group 3 showed no significant change. One case in this group had normal AER at the time of follow-up. At this time group 2 tended to have lower AER than group 1 (P=0.045). Neither HbA1C nor GFR showed significant changes in any of the groups, and as shown in the Table no significant differences were found among the groups during the study period or at follow-up.

Glomerular dimensions
In the baseline biopsies no differences were seen between the three groups except for VPA/GS, which was larger in group 3 (P=0.01 vs groups 1 and 2).

Figures 3Go–5Go show data on VG, VPA and VPA/GS in baseline and follow-up biopsies in the three groups. Glomerular volume and VPA were calculated relative to body surface area, i.e. corrected to a body surface of 1.73 m2. The increase in glomerular volume/1.73 m2 body surface in group 3 was significant (P=0.03). The increase in vascular pole area/1.73 m2 in group 3 almost reached statistical significance (P=0.051) and in group 2 there was a tendency to increase (P=0.07). Vascular pole area, calculated as percentage of glomerular surface, did not show significant change in any group.



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Fig. 3. In each biopsy, the median value, corrected for body surface area, of the 6–26 determinations of glomerular volume is plotted. The increase from baseline (B) to follow-up (F) in group 3 is significant, P=0.03.

 


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Fig. 5. In each corpuscle the ratio of the vascular pole area to calculated glomerular surface is determined and expressed as percentage. The median value in each biopsy is plotted. None of the groups shows significant changes from baseline to follow-up.

 
Glomerular volume, and even more so the VPA, shows a remarkable variation within the space of a renal biopsy, necessitating the fairly large number of corpuscles measured in each biopsy for comparison with follow-up biopsies. In Table 4Go the coefficients of variation are presented. There were no differences among the groups at baseline or at follow-up, except for VPA showing an increase in intra-biopsy variation in the follow-up biopsy in group 3 (29% at baseline and 41% at follow-up). With the number of glomeruli studied per biopsy the coefficient of error was on average <=10% for all the measures. The inter-individual variation within the groups did not decrease after correction for body surface area.


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Table 4. Intra-biopsy variation

 



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Fig. 4. Median vascular pole area, corrected for body surface area, plotted in individual biopsies. The increase from baseline (B) to follow-up (F) in group 3 is close to statistical significance, P=0.051.

 

The cortical interstitium
The measures of cortical interstitial volume fraction are presented in Figure 6Go. The mean values at baseline for the three groups were 20.3%, 22.8% and 25.5%. The differences among the groups did not reach statistical significance (P=0.10). The consistent increase in group 3 (P=0.0009) contrasts to the decrease in group 2 (P=0.03) and the lack of change in group 1.



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Fig. 6. The volume fraction of interstitial space expressed as percentage of tubular cortex increased consistently in all cases in group 3 (P=0.0009) whereas the change over time was not significant in the two anti-hypertensive-treated groups.

 

Correlations among structural changes and structure versus clinical variables
In the baseline data, no correlations nor any tendency to correlations were seen between glomerular volume and glomerulopathy parameters: basement membrane thickness, mesangial volume fraction or the glomerulopathy index (Table 2Go). Neither was there any tendency to correlations between baseline or change in glomerular volume vs change in glomerulopathy parameters during the interval from baseline to follow-up biopsy (data not shown).

The size of the vascular pole area correlated with glomerular volume in baseline biopsies (r=0.74, P=0.0001) as well as in the follow-up biopsies (r=0.81, P<10-4).

Testing relevant correlations between glomerular dimensions and clinical variables the following results were obtained: at baseline no correlation was seen with systemic blood pressure or GFR. HbA1C showed a significant correlation only with VPA/1.73 m2 (r=0.69, P=0.001), whereas the correlation with glomerular volume/1.73 m2 failed to reach statistical significance (r=0.33, P=0.14). Neither glomerular volume nor vascular pole area tended to show differences between female and male patients.

Changes in glomerular dimensions did not correlate with clinical parameters during follow-up, except for VPA which showed a positive correlation with systolic blood pressure (r=0.49, P=0.03).

At baseline positive correlations were observed between interstitial volume fraction and basement membrane thickness as well as matrix parameters in groups 1 and 2 combined, whereas no such correlation was seen within group 3. No correlation was seen in any group between change in interstitial volume fraction vs change in glomerulopathy parameters.

The change in interstitial volume fraction showed a positive correlation with diastolic blood pressure during the study period (r=0.54, P=0.001), whereas no correlation was seen with systolic blood pressure, HbA1C or AER.



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Our study concerns young diabetic patients with increased albumin excretion, predominantly in the low range of microalbuminuria. We choose the lower limit for microalbuminuria at 15 µg/min in overnight urine specimens which is lower than the most frequently used cut-off level of 20 µg/min. However, an excretion of 15 µg/min during night-time corresponds to 20 µg/min in 24-h samples [27]. Including patients with low level microalbuminuria we are dealing with initial stages of structural lesions, e.g. without marked mesangial expansion. Such patients probably are more likely to be amenable to prevention of progression with various treatment strategies. However, it is well known that some of these patients will spontaneously revert to the normoalbuminuric stage [28,29]. The results in the three series of patients that were compared in the present study showed definite differences in that the two groups who had been given anti-hypertensive treatment did not show an increase in glomerular volume or in interstitial volume fraction over the experimental period as compared with a conventionally treated reference group. The observations presented in this paper go in parallel with the electron-microscopy study of the diabetic glomerulopathy, which did not progress over the 40-month experimental period in the anti-hypertensive-treated groups, contrary to observations in the reference group [6]. Also, the structural composition of the juxtaglomerular arterioles showed only a very moderate change in the afferent arterioles [30]. Data from the ESPRIT study has recently appeared in abstract form [7]. Their larger series of anti-hypertensive treated type 1 diabetic patients with nephropathy showed no change in glomerular structure, including glomerular volume, over a 3-year period, irrespective of a clear reduction in urinary albumin excretion in the ACE-inhibitor-treated group, but contrary to our results no progression was observed in the placebo-treated reference group either. Their series at baseline showed a wide clinical range including cases with overt proteinuria.

The most clear-cut result in the present study was the definite increase in interstitial volume fraction in the reference group, whereas no increase was detectable in the anti-hypertensive-treated cases. Although the number of patients is small in the anti-hypertensive-treated groups the substantial increase of 29% in the reference group indicates that we would have been able to detect a similar magnitude of increase with the power of 85% by only including six patients in each group.

It is worth noting that the groups differed in terms of AER during follow-up, and group 2 even tended to reach a lower level than group 1 (Table 3Go). In recent years the hypothesis has been put forward, supported by in vitro studies [31], that proteins leaking from the glomerulus may stimulate tubular production of inflammatory mediators, leading eventually to the increase in interstitial tissue.

The widening of interstitial space seemingly represents accumulation of extracellular material, since the subtraction of capillary space which was also estimated by point counting did not change the result, and cellular infiltrates were not prominent in these cases. Therefore, most likely increased production of extracellular material is the underlying mechanism as also supported by in vitro studies [3133]. In the baseline biopsies the reference group tended to have a higher interstitial volume fraction than the combined groups 1 and 2. Whether this might play a role for the further course cannot be determined. In later stages focal ischaemia due to macroangiopathy may give rise to focal fibrosis with a different distribution pattern and appearance.

The glomerular hypertrophy in diabetes has attracted much attention and this structural change associated with the glomerular hyperfiltration has been hypothesized to be a possible causal factor for ensuing diabetic glomerulopathy [16,34], although it has also been underlined that evidence is non-conclusive as to the pathogenic role [35]. The present data do not permit any firm conclusions as to the question whether early glomerular hypertrophy may play a role in the initiation of diabetic glomerulopathy. At the time of the baseline biopsy diabetic glomerulopathy was already present [22,23,36]. For the elucidation of a possible causal role, information about glomerular volume during the phase of normoalbuminuria would be needed. A problem in comparing glomerular volume and glomerulopathy is the biphasic development in glomerular hypertrophy: the early metabolically determined and partially reversible hypertrophy [37], and a later compensatory phase [35,38,39]. The fact that the present reference group (group 3) showed enlargement of glomerular size over the study period with a concomitant further development of glomerulopathy indicates that these cases may be in the compensatory phase. We did not see correlation between glomerular volume at baseline vs the ensuing development of diabetic glomerulopathy, which does not support the idea of a causative role of glomerular hypertrophy. However, the change in glomerulopathy observed here was quite moderate and the glomerular volume at baseline may already be determined as a combined result of early hypertrophy and initial compensatory size changes.

As to an effect of antihypertensive treatment on glomerular volume, some precautions should be taken. Although glomerular volume did not increase in the treated groups, a treatment effect cannot be substantiated with statistical significance. The increase in group 3, although significant, was small, and the number of cases in the treated groups is therefore too small to detect similar increases with sufficient power. Still, a prevention of glomerular growth in the ACE-inhibitor-treated group is in agreement with experimental data and fits with the growth stimulatory effects of angiotensin II [20,33,40].

The determination of glomerular volume involves methodological problems primarily due to the limited number of corpuscles available in a kidney biopsy. The Cavalieri method by which exact measures of individual glomeruli are obtained has considerable advantage to previously applied methods which included assumptions as to glomerular shape and size distribution [41,42]. In particular, with the Cavalieri method a distribution of glomerular volumes is obtained in each biopsy. The present results demonstrated the very large intra biopsy coefficient of variation, so that a large number of corpuscles must be measured when glomerular size is to be compared in follow-up biopsies.

The estimation of vascular pole area is an approximation as previously discussed [24]. However, increased area in normoalbuminuric and microalbuminuric diabetic patients was clearly demonstrated. The present follow-up study indicated an increase in vascular pole area in parallel with systolic blood pressure. This might be due to the concomitant changes in the wall of afferent arterioles [43] showing an increase in the ratio of matrix vs smooth-muscle cells. This structural change might lead to less protection of the glomerular structures from changes in systemic blood pressure. The size of the vascular pole area, i.e. the lumen of afferent plus efferent arterioles and the intervening cells, is likely to be a determinant factor for the glomerular function. The question of how the measures relate to the in vivo situation cannot be answered, but it seems that the structure surrounded by Bowman's (rigid?) capsule might be fairly resistant to changes during the tissue processing.

Structural data from follow-up biopsies have only been reported in a few studies. To achieve better information on the development of the structural changes in the diabetic kidney such studies seem of great interest. However, they also face some problems in that the follow-up biopsies by necessity are studied at a different point of time, therefore the optimal design of contemporary processing and evaluation of the material is not possible. Further, the question of sampling bias inherent in a kidney biopsy is important. To obtain useful information from such a small sample obviously requires that the parameters under question be fairly uniformly distributed within the kidney. One earlier study dealt with structural parameters in two separate biopsies from individual cases comparing biopsies from right and left kidney taken in cadaveric kidney donors [44]. In these cases glomerular parameters as well as glomerular volume compared well individually in the two biopsies. Noteworthy, much larger interindividual variation was observed in glomerular volume than in glomerular ultrastructure. However, these were normal kidneys whereas we are now dealing with kidney tissue with pathological changes and no exact information is available as to the distribution within the kidney of the early renal lesions in diabetes mellitus. The present study was hampered by the fairly low number of cases in each of the study groups. Further, the use of different embedding media in groups 1 and 2 vs group 3 was an inevitable disadvantage since vestopal is no longer available. A study comparing structural dimensions in parallel embeddings in vestopal and epon had to be given up. When this had been initiated vestopal was no longer available, and our last batch failed to polymerize. Therefore, in the present study, the main emphasis is on comparing the change from baseline to the follow-up biopsy among the three groups.

Altogether the changes in the present groups which were studied over a fairly short interval were modest, but with clear differences between anti-hypertensive-treated groups—whether treated with ACE inhibitor or ß-blocker—and the reference group. The anti-hypertensive-treated patients did not show an increase in interstitial volume fraction or in glomerular volume over the study period. Due to the limited number of patients in each group, and also the non-contemporary reference group the present results are considered preliminary data. These results, however, do give rise to some optimism as to the possibility of interfering with the progression of early stages of diabetic renal lesions, and they therefore call for further studies.



   Acknowledgments
 
Expert technical assistance from Ms Birthe Iversen, Ms Lone Lysgaard, Ms Birtha Saugbjerg, K. Schulttz, G.-M. Taube and T. Jansson is gratefully acknowledged, as is the typing of the manuscript by Ms Karin Ø. Kristensen. This study was supported by grants from Aarhus University Foundation, the Danish Diabetes Association, the Danish Medical Research Council, the Juvenile Diabetes Foundation International no. 190592, Novo Nordic Foundation, the Norwegian Diabetes Association, the Swedish Diabetes Association, the Swedish Diabetes Foundation, the First of May Flower Annual Campaign for Health, the Swedish Medical Association, and the Research Funds of Karolinska Institute.



   Notes
 
Correspondence and offprint requests to: Ruth Østerby, Electron Microscopy Laboratory, Building 03, Aarhus Kommunehospital, DK-8000 Aarhus C, Denmark. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 30. 6.99
Accepted in revised form: 6. 6.00