Diabetologia. Glomerular Size and Structure in Diabetes Mellitus. II. Late Abnormalities. Diabetologia 13, (1977) 9 by Springer-Verlag 1977

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Diabetlgia 13, 43-48 (1977) Diabetlgia 9 by Springer-Verlag 1977 Glmerular Size and Structure in Diabetes Mellitus. II. Late Abnrmalities H. J. G. Gundersen and R.

1 Diabetlgia 13, (1977) Diabetlgia 9 by Springer-Verlag 1977 Glmerular Size and Structure in Diabetes Mellitus. II. Late Abnrmalities H. J. G. Gundersen and R. r Secnd University Clinic f Internal Medicine and the University Institute f Pathlgy, University f Aarhus, ~arhus, Denmark Summary. A study f autpsy kidney material frm six lng-term diabetics and fur cntrls was perfrmed in rder t elucidate the mechanism f the glmerular enlargement in lng-term diabetics. The vlume and the severity f the glmerular lesin were measured in each f a number f randmly selected, pen glmeruli. The relative amunt f slid material was taken as an expressin f the severity f the glmerular lesin. In the lng-term diabetics the vlume f pen glmeruli was almst dubled cmpared t that f cntrls and in the individual subject the enlargement was fund t be inversely related t the relative amunt f slid material in the glmeruli. This indicates that the enlargement f pen glmeruli in lngterm diabetics is due t a cmpensatry hypertrphy rather than t the excessive depsitin f basement membrane material. The number f nuclei per pen glmerulus was increased in lng-term diabetics, but nuclear size was unchanged. Mst f the lng-term diabetics had a large number f ccluded glmeruli, and the individual, relative number f such glmeruli crrelated clsely bth with the duratin f diabetes abve 15 years and the cncentratin f creatinine in serum. It is cncluded that the destructin f glmeruli due t diabetic micrangipathy is cmpensated fr sme years by hypertrphy f the least affected glmeruli. This cmpensatry hypertrphy f glmeruli might well accunt fr the preservatin f renal functin in lng-term diabetics fr a number f years despite the prgressive basement membrane lesins f diabetic micrangipathy. Key wrds: Lng-term diabetes, diabetic glmerulsclersis, diabetic renal disease, glmerular size, cmpensatry glmerular hypertrphy, kidney functin. The develpment f diabetic glmerulpathy starts at the nset f diabetes. By 2 years there is a measurable thickening f the peripheral capillary basement mem- brane [9]. The diabetic micrangipathy prgresses steadily ver the years leading eventually t large accumulatins f basement membrane material within the glmerular tuft, thereby reducing the capillary lumen and the capillary bld flw. n the ther hand, studies f kidney functin have shwn that glmerular filtratin rate (GFR) remains high and unchanged fr many years f diabetes [5], until finally renal insufficiency sets in. The slutin f this paradx might lie in the fact that the glmeruli in lng-term diabetics are larger than nrmal. An increase in glmerular size has previusly been mentined in several descriptins f the diabetic kidney and measurements have verified this bservatin [3, 1]. The glmerular enlargement might represent a cmpensatry hypertrphy f the functining glmeruli, triggered by the lss f thers, by analgy with that seen in ther situatins where a part f the kidney has ceased t functin. Hwever, there is a quite different interpretatin f the glmerular enlargement in lng-term diabetes; the increase in size is thught t be due t an expansin f the tuft because f the accumulatin f slid substances. The aim f the present study was t elucidate the relatinship between the persistently nrmal functin and the prgressive mrphlgical changes f the glmeruli in lng-term diabetes, and in particular t distinguish between the alternative hyptheses regarding the mechanism f the glmerular enlargement. Material and Methds Autpsy material was btained frm 4 nn-diabetics and 6 lng-term diabetics. Pertinent data fr all subjects appear frm Table 1. Renal crtical tissue was fixed in frmalin, embedded in paraffin and cut int sectins at 3 g. The measurements were perfrmed

44 H.J.G. Gundersen and R. sterby: Glmerular Size and Structure II Table 1. Clinical data and the relative number f ccluded glmeruli in lng-term diabetics and cntrls Case n.

2 44 H.J.G. Gundersen and R. sterby: Glmerular Size and Structure II Table 1. Clinical data and the relative number f ccluded glmeruli in lng-term diabetics and cntrls Case n. Age Sex Duratin f ccluded Causes f death diabetes glmeruli years per cent Lng-term diabetics 1 48 M 16 1 crnary cclusin 2 48 M 17 5 cardiac arrest 3 26 F peritnitis 4 39 M cerebral vascular disease 5 38 F pneumnia 6 45 M pneumnia Cntrls 7 51 F < 1 subarachnidal hemrrhage 8 31 M < 1 subarachnidal hemrrhage 9 27 M < 1 cardiac arrest 1 35 F < 1 hanging after staining with haematxylin and esin. All glmerular crss-sectins in which pen capillary lps were visible, were termed "pen", whereas the rest were termed "ccluded". The true glmerular vlume was estimated in the fllwing way: each glmerular crss-sectin was prjected nt a drawing f a series f cncentric circles, thereby permitting allcatin f the class int which it fitted best. n the average 137 (82 t 197) crss-sectins f glmerular tufts were classified in each specimen at a magnificatin f 58 Frm the class-distributin f these crss-sectinal areas the distributin f true vlume f pen and ccluded glmeruli and its mean and variance were calculated accrding t the methd f Saltikv [7]. The percentage f ccluded glmeruli was calculated frm the number f pen and ccluded glmeruli, which was estimated frm the same distributins. The cmpsitin and size f individual glmeruli were determined by a pint cunting prcedure n serial sectins. n the sectin in the middle f the series all pen glmeruti within a pre-selected midcrtical area (29 t 5 g!meruli) were identified and fllwed n every furth sectin in bth directins f the series until disappearance. The measurements were perfrmed at a magnificatin f 9 using a Leitz apchrmatic, aplanatic 4/.74 bjective. The nuclei were cunted and the number f pints falling n nuclei, lumina and the slid part f the glmerular tuft, respectively, was estimated n the individual crss-sectins f all selected glmeruli, emplying a grid with a pint densitiy f 16 pints/1,,u 2. The sum f the three individual fractins - nuclei, lumina and slid - makes up the ttal glmerular tuft. The ttal number f pints falling n every furth sectin f ne tuft is prprtinal t the abslute vlume f that tuft. The fractinal number f pints falling n the slid part f the glmerulus equals the fractinal vlume f slid, which is taken as an expressin f the lng-term diabetic lesin. The directin f the relatinship within each subject between the size f individual glmeruli and their fractin f slid indicates which f the tw befre-mentined mechanism is respnsible fr the glmerular enlargement. Cunting f nuclei and pint cunting the area f nuclear crss-sectins prvides an estimate f the ttal number f nuclei per glmerular tuft and f their mean sectinal area. Thereby it was pssible t estimate whether cellular hyperplasia r hypertrphy was present. The distributin f pints between the glmerular cmpnents was evaluated in pen glmeruli in 3 diabetics (Ns. 1, 2, 3) and 3 cntrls (Ns. 7, 8, 9) by ne technician. In the remaining 3 diabetics and 1 cntrl subject nly nuclear crss-sectins and pints falling n nuclei were cunted. Detailed surveys f the mrphmetric methds and sterelgical principles are given in references flumbers 2 and 8. All abslute vlumes pertaining t each individual were adjusted t a bdy surface f 1.73 m 2, based n a nmgram fr height and weight [1]. Since distributins f vlumes generally are lg-nrmal the cmparisn f vlumes between grups has 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 and simple linear regressin analysis were used in the statistical evaluatin, emplying a 5 per cent limit f significance.

3 H. J. G. Gundersen and R. sterby: Glmerular Size and Structure II 45 % VLUME DISTRIBUTIN F GLMERULI mg/lmt NRMAL z uj n- uj ~ 2-1- Lj % 2~ I, ~ l--- 2' 4 6' PER CENT F GLMERULI CCLUDED Fig. 1. The relatinship between serum creatinine (mg/1 ml) and the relative number f ccluded glmeruli (per cent) in five lngterm diabetics. In the remaining diabetic a recent estimate f serum creatinine was nt available. The cefficient f crrelatin is +.97; 2p =.77. The rdinate is lgarithmic % 1 t ~ DIABEIIC ccluded glmeruli,,,,,m Fig. 2. The vlume distributin f glmeruli in a nrmal subject (upper curve) and in a lng-term diabetic (middle curve: pen glmeruli, lwer curve: ccluded glmeruli). Glmerular crss-sectins in which pen capillary lps were visible, were termed "pen"; the rest were termed "ccluded". The abscissa is lgarithmic Results The relative number f ccluded glmeruli appears in Table 1. Mst f the lng-term diabetics shw a cnsiderably increased number f such glmeruli, and the increase is crrelated bth t the duratin f diabetes (r -=.95, 2p =.39) and t the cncentratin f creatinine in serum (r =.97, 2p =.77, see Fig. 1), the nly parameter f renal functin knwn in these patients. Examples f the calculated distributin f true glmerular vlume are shwn in Figure 2. The individual mean glmerular vlumes in cntrls and lngterm diabetics are shwn in Figure 3. The gemetric mean vlume f pen glmeruli was 1.32 and 2.41 M~t 3 in the tw grups (2p =.1). The gemetric mean vlume f the ccluded glmeruli was 1.71 M~t 3 in the fur diabetics, in whm a large prprtin f such glmeruli was present. The mean size f the ccluded glmeruli was smaller than that f the pen glmeruli in all fur cases. n average their vlume was reduced by per cent (mean and SEM; 2p =.49). The mean area f nuclear crss-sectins was identical in the tw grups, ~t 2 v _ 1.9 ~t 2 (mean and SD), whereas the number f nuclear crss E :D ry.< q k.b LU > M)J ND D D pen ccluded Fig. 3. The mean glmerular vlume (Mp, 3) in fur cntrls (ND) and six lng-term diabetics (D) n a lgarithmic scale. The glmeruli in lng-term diabetics are divided int pen and clsed nes. pen circles cnnected with a line represent the mean vlume f pen and clsed glmeruli, respectively, in the same subject. The hrizntal bars indicate the grup means

46 11-' 1- ~" 9- < UA n,," W n LLI [] 8 7 6-5- i NUCLEAR CRSS SECTINS #2 Z ND < D uj n,,.< Z.< W Fig. 4.

4 46 11-' 1- ~" 9- < UA n,," W n LLI [] i NUCLEAR CRSS SECTINS #2 Z ND < D uj n,,.< Z.< W Fig. 4. The mean number f nuclear crss sectins per pen glmerulus in cntrls (ND) and in lng-term diabetics (D) is given t the left. T the right is shwn the mean sectinal area (~t 2) f the nuclear crss sectins in the same grups. The hrizntal bars indicate the grup means t~ < n- UJ 3s,2- L9 LL UJ 3 > / / 25I ND 9 nrmal diabetic 6~ 7~ l5 ' NUMBER F NUCLEI PER GLMERULUS Fig. 5. Filled and pen circles represent the individual mean number f nuclear crss sectins per glmerulus and mean glmerular vlume (M~t 3) in nrmals and lng-term diabetics, respectively. The line thrugh each circle represents the individual relatinship (the regressin line) between the number f nuclei per glmerulus and the vlume f the glmerulus (pints), based n 29 t 5 glmeruli per individual. The slpe f each line is prprtinal t the individual regressin cefficient in the abve relatinship, whereas the length f the drawn line is arbitrarily fixed D H. J. G. Gundersen and R. ~)sterby: Glmerular Size and Structure II sectins per gimerulus was larger in the diabetics than in the cntrls, v. 61 _+ 18; 2p =.22 (see Fig. 4). Since the mean nuclear crss sectinal area was the same in the tw grups the number f nuclear crss sectins can be taken as an expressin f the ttal number f nuclei per glmerulus in the tw grups. Figure 5 shws the relatinship between the number f nuclei per glmerulus and the vlume f glmeruli. Within each individual the relatinship was statistically highly significant (2p <.1 in all cases), and the mean slpe f the individual regressin lines in diabetics,.279 _+.43 pints/nuclear crss-sectin, was nt different frm that f nrmals,.233 _+.33 pints/nuclear crss-sectin (2p =.11). The relatinship between the number f nuclei per glmerulus and the vlume f glmeruli amng individuals was als statistically significant, r =.8, 2p =.59. Figure 6 illustrates the relatinship between glmerular size and fractin f slid within glmeruli frm ne diabetic subject. It appears that the relatinship is inverse. Figure 7 shws the regressin cefficients (the slpes f the linear regressin lines) frm the three diabetics and the three cntrls. The regressin cefficients were statistically significantly different frm zer in all diabetics but nt in any f the cntrls. Figure 7 als shws that the magnitude f the slpes in diabetics tended t crrelate t the prprtin f ccluded glmeruli, but the number f bservatins is t small t be cnsidered fr statistical analysis. Discussin The results f the present study have shwn that the pen, still functining glmeruli in lng-term diabetics are larger than nrmal. The size f the nuclei is unchanged, but the number f nuclei (an expressin f the number f cells) per glmerulus is increased in prprtin t the increase in glmerular vlume. This might be taken t indicate that the grwth f the glmeruli in lng-term diabetics is brugth abut by cellular hyperplasia rather than hypertrphy. The inverse relatinship between individual glmerular size and the fractin f slid in glmeruli strngly supprts the hypthesis that the enlargement f pen glmeruli is cmpensatry t the lss f the functin f the clsed nes. If the glmerular enlargement were due mainly t depsitin f basement membrane material the situatin wuld have been the ppsite ne: a direct relatinship between size and slid fractin wuld have been fund. Glmerular enlargement has previusly been de-

5 H. J. G. Gundersen and R. sterby: Glmerular Size and Structure II 47 pints 3- ry < 2 W 2- LIJ 2~ 3 I - ~ Y= X 5' 6' 71) RELAIIVE VLUME F SLID, per cent Fig. 6. The relatinship between the relative vlume f slid material (per cent) in individual glmeruli and the vlume f the glmerulus (pints). Each pint represents ne glmerulus fllwed by serial sectining. All glmeruli are frm that lng-term diabetic wh amng the three shwn in Fig. 7 had a median slpe f the regressin line pints/vl~ - Z ~-2 n,, LU n," JT -/, It. LU (D nrmal diabetic lb 2' 3' ~' ' PER CENT F GLMERULI CCLUDED Fig. 7. The rdinate gives the cefficient f regressin between the relative vlume f slid material (per cent) in individual glmeruli and the vlume f glmeruli (pints) in three nrmal subjects and three lng-term diabetics. Nte that the figures n the rdinate are negative. The abscissa gives the relative number f ccluded glmeruli (per cent) in each subject scribed in newly diagnsed diabetics [ 11], in whm an increase f the luminal vlume and the surface area f the capillaries is als fund [4]. In a series f insulintreated shrt-term diabetics (duratin ne t six years) the glmerular enlargement f 25 per cent was nt statistically significant. Hwever, the luminal vlume was still increased in these patients [11]. In cntradistinctin t the present study, n indicatin f cellular hyperplasia was fund in this early phase f the disease. The increase in GFR fllws the same pattern as the enlargement f glmeruli, and it is knwn t remain elevated fr many years f diabetes, decreasing nly when albuminuria begins [5]. The estimates f abslute glmerular vlumes in lng-term diabetics in the present study cannt be cmpared directly t the figures in the abve-mentined study in early diabetes [11] since the latter was made n bipsy material. Such a cmparisn has been made in anther bipsy study [1] revealing an increased glmerular size in lng-term diabetics cmpared t shrt-term diabetics. Therefre, the glmerular enlargement in lng-term diabetics is prbably added t that present already frm the start f the disease. ne rugh mrphlgical measure f this lngterm diabetic kidney disease is the prprtin f clsed glmeruli. Clsure f all capillaries in a glmerular crss sectin is nly the terminal stage f a prlnged prcess during which the thickness f the peripheral basement membrane and the amunt f basement membrane like material in the mesangium is steadily increasing. Therefre, the very clse crrelatin between the duratin f diabetes abve apprximately 15 years and the prprtin f clsed glmeruli des nt mean that the lng-term diabetic glmerulpathy starts at this time. It seems mre reasnable t translate this crrelatin int the statement that it takes abut 15 years fr the micrangipathy t reach the terminal stage in single glmeruli. Frm this time the demand n the rest f the glmeruli t increase their functin and size is likely t be mre and mre imperative. Hwever, since the number f functining glmeruli cntinues t decrease, the final utcme fr the kidney as a

6 48 H. J. G. Gundersen and R. sterby: Glmerular Size and Structure II whle is cessatin f functin, unless means t delay the destructive prcess can be fund. Lwering f the bld pressure is ne way which is being studied at present [6]. Frm the results presented in this study we cnclude that kidney functin in lng-term diabetics remains abve critical limits due t a cmpensatry grwth f sme glmeruli which fr a certain perid balances the destructin f thers. Acknwledgments. We are very much indebted t Dr. M. Gregersen, Department f Frensic Medicine, fr the nrmal kidney tissue. We als want t thank Mrs. Drte Eilertsen, Mrs. Grethe Glerup, Mrs. Helen Hu Jrgensen and Mr. Erik Muritzen fr skilful technical assistance. The study was supprted by grants frm Statens lzegevidenskabelige Frskningsr~d, Nrdisk Insulinfnd, the University f Aarhus, P. Carl Petersens Fnd, and Landsfreningen fr Sukkersyge. References 1. Diem, K., Lentner, C.: Scientific tables. Basel: Geigy Elias, H., Hennig, A., Schwartz, D. E.: Sterelgy: applicatins t bimedical research. Physil. Rev. 51, (1971) 3. Klein, L., Butcher, D. L., Sudilvsky,., Kikkawa, R., Miller, M.: Quantificatin f cllagen in renal glmeruli islated frm human nndiabetic and diabetic kidneys. Diabetes 24, (1975) 4. Krustrnp, J. P., Gundersen, H. J. G., r R.: Glmerular size and structure in diabetes mellitus. III. Early enlargement f capillary surface. Diabetlgia (in press) 5. Mgensen, C. E.: Kidney functin and glmerular permeability t macrmlecules in juvenile diabetes. Dan. med. Bull. 19 (Suppl. 3), 1-38 (1972) 6. Mgensen, C. E.: Renal functin changes in diabetes. Diabetes 25, (1976) 7. Saltikv, S. A.: The determinatin f the size distributin f particles in an paque material frm a measurement f the ~ize distributin f their sectins. In: Sterelgy (ed. H. Elias), pp Berlin: Springer Verlag Weibel, E. R.: Sterelgical principles fr mrphmetry in electrn micrscpic cytlgy. Int. Rev. Cytl. 26, (1969) 9. sterby, R.: Early phases in the develpment f diabetic glmerulpathy. A quantitative electrn micrscpic study. Aeta med. scand. Suppl. 574, 1-82 (1975)!. sterby, R., Gundersen, H. J. G., Mgensen, C. E.: Increased glmernlar size in shrt-term and lng-term juvenile diabetics. Excerpta Medica, Internatinal Cngress series n. 28, p. 191, Amsterdam sterby, R., Gundersen, H. J. G.: Glmerular size and structure in diabetes meuitus. I. Early abnrmalities. Diabetlgia 11, (1975) Received: August 2, 1976, and in revised frm: ctber 15, 1976 ~ Dr. R. sterby Department f Pathlgy Kmmunehspitalet DK-8/?~a'hus C Denmark

Glomerular Size and Structure in Diabetes Mellitus I. Early Abnormalities

Glomerular Size and Structure in Diabetes Mellitus I. Early Abnormalities 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