Diabetlgia 11, 225--229 (1975) 9 by Springer-Verlag 1975 Glmerular Size and Structure in Diabetes Mellitus I. Early Abnrmalities R. 0sterby and H. J. G. Gundersen University Institute f Pathlgy and the Secnd University Clinic f Internal Medicine, Kmmunehspitalet, Aarhus, Denmark Received: December 9, 1974, and in revised frm: March 19, 1975 Summary. The vlume f the glmerular tuft and its cmpnents was estimated in juvenile diabetics at nset and after ne t six years' duratin f the disease. An enlargement f the glmeru!ar tuft was demnstrated in the newly diagnsed diabetics. In these patients the vlume f bth the individual capillary lumina and f the individual glmerular cells was enlarged, whereas the number f ceils was unchanged. Althugh a partial nrmalizatin is seen in diabetics after a few years' disease, such patients still have an elevated capillary vlume. The pssible relatinship between these findings and the well-dcumented elevatin f the glmerular filtratin rate in diabetics is discussed. It is suggested that the mechanism behind the mrphlgical and functinal changes is either a pressure-induced unflding f the capillary wall r an increase f the capillary wall area, but the present study permits n distinctin between these pssibilities. Key wrds: Juvenile diabetes, early diabetes, glmerular hypertrphy, glmerular mrphlgy. The glmerular filtratin rate (GFR) is increased in juvenile diabetics at the very beginning f the disease [6], and the elevatin f the GFR persists during the first decade [3, 7, 14]. The mechanisms behind this functinal abnrmality are prly understd, but it seems mst likely that the functinal defect is lcalized in the glmerulus and nt in the ensuing part f the nephrn [8]. The present investigatin was therefre undertaken in rder t study the size and mrphlgy f the renal glmerulus in early diabetes. Material The cntrl grup cnsists f 8 subjects (see Table 1) withut clinical and labratry evidence f diabetes r renal disease. Tw grups f juvenile diabetics were investigated, ne grup at the acute nset f disease r within three t six weeks f treatment with insulin and diet (referred t as newly diagnsed diabetics), the ther grup after ne t six years f treatment (referred t as shrt-term diabetics), see Table 1. A quantitative study f the glmerular ultrastructure in the same patients has been presented elsewhere [16]. Frm the newly diagnsed diabetics material fr light micrscpy was available frm bipsies btained bth befre and a few weeks after initiatin f treatment. It was decided t carry ut all f the investigatins n the first bipsy, if pssible, but the quantitatin f glmerular structures described belw requires sectins that are f high quality cncerning fixatin, staining and sectining. Cnsequently, if the bipsy btained befre treatment was nt useful, the bipsy btained after initiatin f treatment was used. The studies mentined abve [16] indicated that the ultrastructure f the renal glmerulus is the same in the untreated state and after three t six weeks f treatment. Methds 1 Percutaneus needle bipsies f the kidney were btained with the teclmique intrduced by Brun and Raaschu [1]. The bipsies were perfrmed ver a perid f fur years in randm rder in the three grups cnsidered. A small piece f tissue was cut frm either end f the bipsy and was fixed fr electrn micrscpy. The rest f the cylinder was fixed in either phsphate-buffered frmalin r in Helly's fluid, embedded in paraffin and cut parallel t the lng axis int serial sectins at 3 ix. The measurements were perfrmed n PAS-stained sectins, spaced mre than 200 9 apart. Fr the determinatin f glmerular size additinal sectins were used in sme cases. In the fllwing 'glmerulus' means the glmerular tuft, and therefre des nt include the capsule f Bwman and the capsular space. 1 Detailed surveys f the emplyed mrphmetric methds and sterelgical principles are given in references numbers 5 and 15.
226 R. sterby and H. J. G.: Gundersen: Glmerular Size and Structure All measurements were perfrmed in ne run by the same technician wh was unaware f the identity f the specimens. Preliminary studies had shwn that the glmeruli btained frm percutaneus needle bipsies fitted best t rtatinal ellipsids with a linear excentricity f 0.8 and with the lng axis parallel t the needlebipsy. sectinal areas frm a rtatinal ellipsid, which has been cut parallel t the lng axis, equals that f a sphere. The calculatin f the mean glmerular vlume was based n an average f 66 glmerular crss sectins frm each bipsy. The nuclei within the glmerular tuft were cunted and the relative areas f the lumina, the nuclei and the remaining slid part f the glmerulus, (i.e. gl- Table 1. Clinical data and the number and mean area f glmerular nuclei (adjusted t a bdy surface fl.73 m 2) in the cntrls and the tw grups ] diabetics Age Sex Bdy Duratin Nuclei Mean nuclear surface, m e f diabetes per mean area ~t 2 years glmerular crss-sectin 16 F 1.49 117 39 17 F 1.83 50 25 20 F 1.77 82 38 28 F 1.73 100 26 31 F 1.32 103 35 12 M 1.44 116 24 19 M 2.04 98 41 22 M 1.86 83 15 Mean 21 1.69 94 30 SD 6 0.24 22 9 14 F 1.38 0 120 33 ~~ 16 F 1.49 0 116 52.~ 18 M 1.84 0 84 28 "~ ~ 19 M 1.76 0 83 26 ~ h3 26 M 1.80 0 75 40 2; 32 M 1.81 0 130 42 Mean 21 1.68 101 37 SD 7 0.19 23 10 20 F 1.66 4.5 99 30 17 M 1.83 5.0 112 26 17 M 2.04 5.0 107 25, 22 M 1.72 1.5 123 31 23 M 1.66 6.0 94 31 27 M 1.85 4.0 74 23 r~ ~ 28 M 2.08 1.0 71 29 28 M 1.80 3.5 60 19 31 M 1.98 4.5 63 23 32 M 1.57 1.0 -- -- Mean 25 1.82 3.6 89 26 SD 6 0.17 1.8 23 4 Frm all slides each f the glmerular crss-sectins was prjected nt a drawing f a series f cncentric ellipses, thereby permitting a decisin f the class int which it fitted best. Frm the class-distributin f these randm crss-sectinal areas the distributin f true glmerular vlume and its mean value were calculated accrding t Saltikv [12], taking int cnsideratin that the distributin f crss- merular cells f all types and the extracellular material), were estimated by pint-cunting. A ttal f apprximately 1000 pints was cunted in each bipsy. This was dne at a magnificatin f 900 emplying a grid with a pint density f 16 pints/10 000 ~t 2 and a Leitz apchrmatic, aplanatic 40/0.74 bjective. Since the relative vlume f a structure in space equals its relative area n sectin, the abslute vlume
R. sterby and H. J. G.: Gundersen: Glmerular Size and Structure 227 f a structure in an average glmerulus was btained by multiplying the relative area f the structure by the mean glmerular vlume. Finally, all lumina larger than 10 F 2 n sectin were cunted, emplying a grid with a pint density f 2 pints per 92. The number f such large capillaries in an equatrial crss sectin f a spherical, imaginary glmerulus f average size (termed 'mean glmerular crss sectin') was then calculated fr each bipsy. The bdy surface f each subject was read ff in a nmgram based n weight and height [2]. All individual values fr abslute vlumes r areas f the glmerulus and its structures were then adjusted t a standard bdy surface f 1.73 m-7 Since distributins f vlumes generally are lgnrmal the cmparisns f vlumes between grups have been carried ut n the lgarithmically transfrmed values. The mean values calculated frm these distributins equal the gemetric means f the riginal values. Student's t-test was emplyed in the statistical treatment, using a 5Y limit f significance. Results The vlume f the glmerular tuft in the cntrls and the tw grups f diabetics is shwn in Fig. 1. The gemetric mean glmerular vlume in the newly diagnsed diabetics was 1.41 M~0 (millin Vs) whereas the cntrls had a gemetric mean f 0.83 M~0. The difference between these tw values is statistically significant, 2 p = 0.014. The diabetics with ne t six years' duratin f disease had a gemetric mean f 1.05 M~P. This value was nt significantly different frm either that f the nrmals r that f the newly diagnsed diabetics. (The crss-sectinal diameters crrespnding t the glmerular vlumes mentined abve are 139, 117 and 126 ~t). By the quantitatin f the glmerular structures (capillary lumina, slid parts, nuclei) it was pssible t determine which part f the glmerulus that was invlved in the changes just mentined. The vlume f capillary lumen per glmerulus in the newly diagnsed diabetics was almst twice that f the cntrls, the gemetric mean values were 0.38 M98 and 0.20 MF8 respectively, 2 p = 0.0086 (Fig. 1). Likewise, the number f large capillaries per mean glmerular crss sectin was dubled in the newly diagnsed diabetics, wh shwed a gemetric mean value f 5.21 cmpared t 2.82 in the cntrls, 2 p = 0.014 (Fig. 2). The slid vlume per glmerulus was als enlarged in the newly diagnsed diabetics, 0.84 M~t 3 versus the cntrl value f 0.51 M~P, 2 p = 0.021 (Fig. 1). n the ther hand, the number f cells per glmerulus was the same in the tw grups, expressed as the number f nuclei per mean glmerular crss sectin, see Table 1. It is ntewrthy that within the grup f newly diagnsed diabetics a statistically significant relatinship existed between the individual mean nuclear area M)J 3 3.2-1.6. 0.8-0.4-0.2-0.1 GLMERULUS LUMINA SLID li! " I 41- M~ 3.3.2 1.6 8 ++.+. + I. 0.+,. ~.0.t, C De Dr6 C D +- C D DvG Fig. 1. Vlumes f the ttal glmerular tuft, f the capillary lumen and f the slid part f the glmerulus. The vlumes are given n an lg scale in millins f ~3 per 1.73 m 2 bdy surface. C: cntrls, D: newly diagnsed diabetics, DI_G: patients with 1--6 years' duratin f diabetes. pen circles: females, filled circles: males. The hrizntal bars indicate mean values 16. < 8- Z ~r --3 --J W n,, --I 0 W 0 I z 2-9 0 9 $ 8 C D D'1-6 Fig. 2. Capillary lumina larger than 10 ix 2 n sectin. The number per mean glmerular crss sectin is shwn n a lg scale. Symbls as in Fig. 1 9.0.2 "0.I
228 R. sterby and H. J. G.: Gundersen: Glmerular Size and Structure n the ne hand and the vlume f the whle glmerulus (r = 0.87, 2 p = 0.025) and that f the slid part (r = 0.86, 2 p = 0.027) n the ther hand (Fig. 3), whereas nne f these relatinships were present ha the cntrl grup r the grup f shrt-term diabetics. The mean nuclear area in the newly diagnsed diabetics, hwever, was nly slightly and statistically insignificantly greater than that f the cntrls, see Table 1. The study f individual glmerular structures in the shrt-term diabetics revealed that nly slight and statistically insignificant regressin f the changes fund in the newly diagnsed diabetics had ccurred (see Fig. 1 and 2). It is f special interest that the vlume f glmerular capillary lumen in the shrtterm diabetics was still increased cmpared t that f the nrmals, 0.31 M~0 and 0.20 MgZ, respectively, 2 p = 0.021. -6 M lfl : 1.50" 1.25, t 0.75 0.50 llll II 2 ~ A 6 6b,2 Mean nuclear area Fig. 3. The relatinship between the mean nuclear area and the vlume f the slid part f the glmerulus in the newly diagnsed diabetics (r = 0.86, 2 p = 0.027) As in the newly diagnsed diabetics bth the number f cells per glmerulus and the nuclear area were nrmal in the shrt-term diabetics (see Table 1), but a cmparisn between the tw grups f diabetics did demnstrate a decrease in nuclear area, 36.9 ~t ~ versus 26.3 ~t ~, 2 p = 0.046. Discussin The demnstrated deviatins frm the nrmal f the varius measures f the glmerular tuft in newly diagnsed diabetics mean that mrphlgical changes f the first part f the nephrn are present cncmitant with the acute functinal changes f the same part f the nephrn, i.e. the increased glmerular ill- it tratin rate. The rentgengraphic demnstratin f a 20y enlargement f the whle kidney in early diabetes [10] furthermre indicates that changes are simultaneusly present in ther segments f the nephrn, since the glmeruli cnstitute nly abut 2Y f the ttal vlume f the kidney [4]. The measurements f the individual glmerular structures shwed that bth the slid part and the capillary lumen was enlarged. In a light micrscpic study it is nt pssible t distinguish between the cytplasm and the basement membrane material in the slid part f the tuft. Hwever, quantitative electrn micrscpic studies [16] have indicated that bth the amunt f mesangial basement membrane material and the thickness f the peripheral basement membrane are nrmal in newly diagnsed diabetics. The increased vlume f the slid part f the glmerular tuft in cmbinatin with the unchanged number f nuclei per glmerulus means that the vlume f the individual cell is increased, i.e. hypertrphy is present in diabetics. The same cnclusin can be drawn frm the psitive crrelatin between slid vlume and mean nuclear area in the individual newly diagnsed diabetics (Fig. 3). Furthermre, results frm studies f acute diabetes in rats shw that bth the RNA/DNA-rati [13] and the ttal prtein cntent [11] are increased in the kidney as a whle. It is nt knwn t which extent these findings are valid fr the glmerulus. The increased capillary vlume per glmerulus fund in bth grups f diabetics in the present study culd well be the mrphlgical change underlying the elevated glmerular filtratin rate (GFR) in these patients. This elevatin f the GFR, which has cnsistently been demnstrated in diabetics, bth at nset and during the first decade f the disease [8], has furthermre been shwn t be mst prnunced in newly diagnsed patients, wh als shwed the largest capillary vlumes. The mechanism behind this enlargement culd either be a) increased pressure within the capillaries leading t an unflding f the capillary wall r b) cellular hypertrphy resulting in an increase f the capillary wall area. Theretically, an increase in the ttal capillary vlume culd be due either t an increase in the crsssectinal area f the individual capillaries f the glmerulus r t an increase in the number f capillaries per glmerulus. Hwever, the finding f an unchanged number f nuclei, but an increase in number f capillaries with large crss-sectinal areas, shws that the increase in ttal capillary vlume in newly diagnsed diabetics is due t an increase in the size f individual capillaries.
R. sterby and H. J. G.: Gundersen: Glmerular Size and Structure 229 Bth f the mechanisms prpsed abve fr the increase in capillary vlume wuld result in an increased GFR, due either t the elevated pressure gradient acrss the filtratin barrier r t the enlargement f the ttal filtratin surface. Hwever, the distinctin between the tw pssibilities -- either a pressure-induced unflding r an enlargement f the capillary wall area -- as the cause f the increase in capillary vlume is nly pssible by direct mrphmetric determinatin f the ttal capillary surface. Such measurements (the number f intersectins between the surface and a test-line) require a very sharp demarcatin f the luminal brder f the capillary wall, which is nly btainable in plastic-embedded bipsy specimens. The material available cnsisted f sectins frm paraffin-embedded bipsies, and thus the requirement mentined abve culd nt be fulfilled. It therefre remains an pen questin which mechanism is respnsible fr the increase in capillary vlume (and the increase in GFR). It is ntewrthy that all estimates f vlumes in the shrt-term diabetics are clser t nrmal than thse f the newly diagnsed diabetics, but that the capillary vlume is still elevated. This is in gd agreement with the finding f a decrease in bth the lddney size and GFR during the first few mnths f insulin-treatment f the metablic disturbance [9, 6], thugh the GFR is nt cmpletely nrmalized [6]. It is nt knwn fr hw lng the described mrphlgical changes persist after the first few years cnsidered in the present study. The GFR, hwever, remains elevated in lng-term diabetics, irrespective f the duratin f the disease, as lng as prteinuria has nt ccurred [7]. It is, therefre, likely that the enlargement f the glmeruli and f their capillaries and cells, which is present frm the beginning f the disease, als persists fr many years. It is, hwever, difficult t substantiate this prpsal in lng-term diabetics due t the very cmpsite mrphlgical picture in these patients, which is characterized t a large extent by the advanced basement membrane lesins f diabetic angipathy. ne element f this picture is a prnunced enlargement f the still functining glmeruli, which is prbably related t the simultaneus cclusin f ther glmeruli due t the excessive accumulatin f basement membrane material (Gundersen, H. J. G., sterby, R.: unpublished bservatins). Acknwledgements. We are very much indebted t Drs. G. Gregersen, B. Mller and H. Skjldbrg fr the kidney bipsy material. We wish t thank Mrs. Drte Eilertsen, Mrs. Grethe Glerup and Mrs. Helen Hu Jrgensen fr their skilful assistance. The study was supprted by grants frm Nrdisk Insulinfnd. References 1. Brun, C., Raaschu, F.: Kidney bipsies. Amer. J. Med. 24, 676--691 (1958) 2. Diem, K., Lentner, C.: Scientific tables. Basel: Geigy 1970 3. Ditzel, J., Schwartz, M.: Abnrmally increased glmerular filtratin rate in shrt-term insulin-treated diabetic subjects. Diabetes 16, 264--267 (1967) 4. Elias, H., Hennig, A.: Sterelgy f the human renal glmerulus. In: Quantitative methds in mrphlgy (eds. E. Weibel, H. Elias) pp. 130-166. Berlin: Springer Verlag 1967 5. Elias, H., Hennig, A., Schwartz, D. E.: Sterelgy: Applicatins t bimedical research. Physil. Rev. 51, 158--200 (1971) 6. Mgensen, C. E.: Kidney functin and glmerular permeability t macrmlecules in early juvenile diabetes. Scand. J. clin. Lab. Invest. 28, 79--90 (1971) 7. Mgensen, C. E.: Glmerular filtratin rate and renal plasma flw in shrt-term and lng-term juvenile diabetes mellitus. Scand. J. clin. Lab. Invest. 28, 91-- 100 (1971) 8. Mgensen, C. E.: Kidney functin and glmerular permeability t macrmlecules in juvenile diabetes. (Thesis). Dan. med. Bull. 19, suppl. 3, 1--38 (1972) 9. Mgensen, C. E., Andersen, M. J. F.: Increased kidney size and glmerular filtratin rate in untreated juvenile diabetes: Nrmalizatin by insulin treatment. Diabetlgia 11, 221--224 (1975) 10. Mgensen, C. E., Andersen, M. J. F.: Increased kidney size and glmerular filtratin rate in early juvenile diabetes. Diabetes 22, 706--712 (1973) 11. Rss, J., Gldman, J. K.: Effect f streptztcin-induced diabetes n kidney weight and cmpensatry hypertrphy in the rat. Endcrinlgy 88, 1079-- 1082 (i971) 12. Saltikv, S. A.: The determinatin f the size distributin f particles in an paque material frm a measurement f the size distributin f their sectins. In: Sterelgy (ed. H. Elias) pp. 163-- 173. Berlin: Springer Verlag 1967 13. Seyer-Hansen, K.: Renal hypertrphy in experimental diabetes. (Abstract) Diabetlgia 10, 386--387 (1974) 14. Stalder, G., Schmid, R., v. Wlff, M.: Funktinelle Mikrangipathie der Nieren beim behandelten Diabetes mellitus im Kindesalter. Dtsch. reed. Wschr. 85, 346--350 (1960) 15. Weibel, E. R.: Sterelgical principles fr mrphmetry in electrn micrscpic cytlgy. Int. Rev. Cytl. 26, 235--302 (1969) 16. sterby, R.: Early phases in the develpment f diabetic glmerulpathy. A quantitative electrn micrscpic study. Acta reed. Scand., suppl. 574, 1975 R. sterby, M.D. Dept. f Pathlgy Kmmunehspitalet DK-- 8000 Arhus C Denmark