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
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Abstract |
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Methods. Microalbuminuric type 1 diabetic patients with diabetes duration of 616 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
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Introduction |
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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.
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Subjects and methods |
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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 1 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 2
), 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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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 300400 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 010 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 912 in the groups (range 626). The volume of individual glomeruli is: t*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 1). The size of VPA was calculated as the sum of chords factored by the distance between sectioning levels.
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Interstitial volume fraction
For this estimate five to eight 1-µm sections were measured by point counting in each biopsy (Figure 2). 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 1.67.9x106 µm2).
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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 KruskalWallis test. When this test showed significant differences, results in groups were compared with the aid of the MannWhitney two-sample test. Correlations were studied by simple least-squares regression.
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Results |
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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 35
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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The cortical interstitium
The measures of cortical interstitial volume fraction are presented in Figure 6. 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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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 2). 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.
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Discussion |
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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 3). 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 groupswhether treated with ACE inhibitor or ß-blockerand 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.
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Acknowledgments |
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Notes |
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References |
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