The Physiological and Pathophysiological Basis of

Schurek et al.: Pathophysiology of glomerular permeability

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Eur. J. Clin. Chem. Clin. Biochem. Vol. 30, 1992, pp. 627-633 © 1992 Walter de Gruyter & Co. Berlin · New York

The Physiological and Pathophysiological Basis of Glomerular Permeability for Plasma Proteins and Erythrocytes By H.-J. Schurek'\ K.-H. Neumann, H. Flohr, M. Zeh and H. Stolte Medizinische Hochschule Hannover, Zentrum Innere Medizin, Abt. Nephrologie, Hannover, Germany (Received March 4, 1992)

Summary: The barrier function of glomerular capillaries in vivo, which prevents the leakage of plasma proteins and cellular elements, depends on the basic morphological and electro-chemical fine structure of the glomerular capillary wall, and on a functional barrier maintained by components obtained from blood, which effect the definitive barrier against the leakage of plasma proteins and cellular elements. The functional component of the barrier may explain the variability and some of the phenomena known as functional proteinuria. A certain size and number of morphological "defects" are thought to represent the normal condition, but under pathological conditions they may increase in size and number, resulting in a shift to an increasing permeability for higher molecular mass proteins; also an increase of the size and number of larger defects may enable more red cells to pass the barrier compared with the normal condition. These defects are different from the minimal glomerular lesions which are due to charge defects in the glomerular capillary membrane, primarily the lamina rara interna and the lamina rara externa of the basement membrane.

1. Glomerular Capillaries' Barrier Function in Vivo

The glomerular capillary wall in the kidney exhibits distinct properties compared with other tissue capillaries. For example, water permeability is quite high and the permeability for plasma proteins is quite low. If one uses experimental data (early proximal micropun ture samples, (1, 2)) from rat kidneys to calculate filtered protein in man, the result is 1.8 g albumin glomerularly filtered from 180 litres of ultrafiltrate per day. Most of the filtered albumin and low molecular mass proteins and peptides are reabsorbed during passage along the proximal tubules (3). Thus, only trace amounts of low molecular mass proteins, peptides and some 20 to 40 mg of albumin are found in the end urine. High molecular mass proteins are practically not filtered under physiological conditions. High molecular mass proteins, which can be found in ') Funding: Deutsche Forschungsgemeinschaft Grant Schu 343; Deutsche Forschungsgemeinschaft SFB 146. Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 10

trace amounts in the end urine are thought to be released from distal tubular segments and the lower urinary tract as Tamm-Horsfall protein and secretory IgA. 1.1 Hydraulic conductivity Access to the filter is controlled by the size and number of the endothelial fenestrae, which expose 30 — 40% of the glomerular basement membrane (4). Water filtration may be so high because its extracellular route bypasses hydrophobic lipid layers such as cell membranes. The main resistance for water filtration is provided by the slit pores of epithelial foot processes (5). Their morphology was first described in 1974 by Rodewald & Karnovsky (6). Slit surface is only 2 — 3% of the capillary surface (7). The hydraulic conductivity seems to be not only a passive resultant of physical forces; it can also be varied by multiple modifiers (8), not least by changes of the geometry of slit pores (5).

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1.2 Permability of macromolecules, selectivity of size and charge The sieving characteristics of glomerular capillaries for plasma proteins are determined mainly by two attributes: selectivity for molecular size and charge at physiological pH. Size selectivity is determined by the collagen structure of the glomerular basement membrane especially its middle layer, the lamina densa, the main component of which is collagen type IV (9). The collagen structure of the lamina densa is sufficiently wide-meshed to allow passage of 20% of electrically neutral chain molecules (dextrans) with the molecular mass of albumin (Stoke's radius of 3.6 nm), i.e. a sieving coefficient of 0.2 (10). Macromolecules ranging in size from 2—4 nm (20—40 A) are discriminated, passage of the smaller molecules being high and that of the larger molecules being low. The reason for the poor passage of albumin (sieving coefficient 0.3 χ 10~3) may be it's lower deformability and stretching properties compared with chain molecules (11) and the basal negative electric charge at the surface of this molecule and of other plasma proteins. At physiological pH this allows repulsion between the molecules and their good solubility, but it also results in repulsion at the negatively charged filtration barrier of the glomerular capillaries. Both endothelium and epithelium are coated by a strongly negatively charged glycocalix (sialic acid containing glycoproteins) at the cell surface. Moreover in front of and behind the lamina densa of the glomerular basement membrane there is a negatively charged layer, the lamina rara interna and externa, consisting mainly of hyaluronic acid glucosaminoglycan molecules (12). By using monocationic dyes it has been demonstrated (13, 14) that the electrostatic meshes are so tightly arranged that this may be explain the low albumin leakage into the glomerular basement membrane (0.03% of plasma concentration within ultrafiltrate). Whether other charged molecules play a significant role has to be established. The lamina rara externa, which embodies the foot processes of epithelial cells, exhibits charged mesh similar to that of lamina rara interna. This secondary charge barrier may slow down the passage of polycationic proteins, which may explain pinocytotic vacuoles within epithelial and mesangium cells (11). Proteins of the size of albumin may pass slit pores directly (slit pore size 4 x 1 4 nm, (6)). Penetration of larger macromolecules has been shown to be dependent on their surface charge. Cationic macromolecules penetrate as far as the epithelial slit pores, where they are rejected, if their steric size is significantly above the size of albumin (15, 16). Anionic macromolecules are more or less rejected at the lamina rara interna, thus prevent-


ing their penetration into the lamina densa. The analysis of the sieving of size equivalent dextran molecules of different charge exhibits large differences of glomerular permeability (10). The discriminating size range of negatively charged macromolecules is more or less 3—4 nm (30—40 A) of Einstein-Stoke's radius. 1.3 The haemodynamic effect on permeability

protein

Ryan & Karnovsky demonstrated (17) that it is important to analyse glomerular barrier function during intact haemodynamics, i. e. by in situ fixation (dripping fixation) of glomeruli at the renal surface. When the haemodynamics are disturbed in parallel with a reduced glomerular filtration rate, penetration of plasma proteins is found to be enhanced; in the case of albumin complete passage through the barrier has been shown (enhanced diffusion with reduced convection). Permeability for macromolecules can be increased at an elevated hydrostatic pressure in glomerular capillaries, i. e. experimentally this has been shown under the influence of angiotensin II; glomerular albumin permeability has been shown to increase significantly (18, 19). Under these conditions, a change of size selectivity of glomerular capillaries for the passage of dextran molecules (20), with enhancement of the permeability for higher molecular mass dextrans has been shown. In parallel water conductivity has been demonstrated to decrease (21), possibly due to mesangial cell constriction or a changing geometry of epithelial slit pores accompanied by vasoconstriction of the efferent arteriole. Also, the permeability for macromolecules was shown to be increased (18) during an excess of catecholamines (norepinephrine, epinephrine), i. e. in cardiac failure (22, 23). Feld et al. (24) have demonstrated differences of glomerular permeability for macromolecules between surface and juxtamedullary glomeruli, using a rat strain with spontaneous hypertension. The most likely reason for this higher permeability of glomeruli of the inner cortex is their short forerunning interlobular artery, which, since the constrictable artery segment is short, may be less effective than surface glomeruli in preventing hypertensive stress. In this context it is worth mentioning that after unilateral nephrectomy, a small but significant increase of filtered plasma proteins has been found in the end urine in man (25). As glomerular haemodynamics as well as filtration dynamics are dependent on the interplay of multiple hormones under physiological conditions, a transitory increase of glomerular permeability is plausible, but it can be found in end urine only under well defined Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 10


conditions (22, 26). A variety of clinical syndromes can be ascribed to this functional increase of glomerular protein permeability, such as orthostatic proteinuria, march proteinuria, proteinuria of heart failure, elevated renal venous pressure or proteinuria of fever. Elevated glomerular capillary pressure may be a common denominator under these conditions. Ryan & Karnovsky (17) tried to explain these phenomena by an accumulation of macromolecules in front of the separation membrane. One of the problems of this hypothesis is that it does not easily explain all the experimental data. An alternative explanation is given in section 3.0.

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leaks of glomerular capillaries, since they are equivalent to penetration of each glomerulus by 1 erythrocyte p?r day, or penetration one in 50 glomeruli by one cell per 30 min, the others being non-permeable. In special forms of glomerulopathy, however, it has been possible to demonstrate these defects, especially in the acute phase of the disease with macrohaematuria, as in IgA nephropathy, where the reduced stability of glomerular capillaries at the locus of immune deposits leads to defects punched out of the glomerular basement membrane (34). This observation may be of special interest, because of a parallel leakage for plasma, which is thought to damage tubular epithelial cells, when it is presented from the tubular 2.0 Pathology of the Barrier Function of Glomerular lumen (35) by complement-induced cell lysis. Phases of plasma leakage may be short pulses, in the short Capillaries time interval between cell passage. Also, in other 2.1 Charge selectivity forms of glomerular lesions associated with microWhile permeability to protein may be enhanced by haematuria, discontinuities of the glomerular capilhaemodynamic factors, it may be very much enhanced lary wall have been demonstrated that are large in glomerulonephritis by pathologic processes directly enough to permit the passage of erythrocytes after active at the glomerular capillaries. The main and deformation (36, 37). It is known (36), that a defect most likely defect of glomeruli in the "minimal lesion" of 250 nm in diameter is large enough for the passage is a rarefication of the charge mesh of the glomerular of erythrocytes (diameter 7 μιη). The relatively large capillaries (27, 28), together with a reduction of the capillary wall defects, which may explain micro- or density of "pores", characterized by a shift of molec- macrohaematuria, may be too small in number to ular sieving curves for neutral dextrans to the left; enhance the accompanying proteinuria significantly, this results in a selective manifold increase of the as the defect may be blocked nearly all the time by permeability for albumin. As the lamina densa is penetrating cells. The recently described leakage for intact, larger macromolecules are rejected to the ex- very large molecules like oc2-macroglobulin into the tent that albumin is the dominant protein in end urine urine (38) has been used as a diagnostic marker, not ("selective proteinuria"). Additionally parts of the for glomerular leakage but for postrenal haematuria; glycocalix are lost and thereby foot processes of epi- this molecule does not penetrate glomerular capillary thelial cells fuse, and slit pores are lost. This results walls under normal conditions, or in different forms in a reduced hydraulic permeability and consequently of glomerulonephritic kidneys. In glomerular diseases, a reactive elevation of the glomerular capillary pres- such as membraneous glomerulopathy, it has been sure (as filtration no longer occurs extracellularly demonstrated, using dextran clearances (39), that the through filtration slit pores but through the epithelial permeability of dextrans with an Einstein-Stoke*s radius < 3 nm is reduced due to a reduction of the cells themselves). number of standard pores. However, the permeability 2.2 Size selectivity and passage of eryth- of larger dextran molecules (Einstein-Stoke'§ radius > 4 nm) is due to the development of membrane rocytes defects large enough for the penetration of larger In composite forms of glomerular inflammatory re- macromolecules, which may explain the pattern of actions cells may invade, cytokines may induce focal „unselective" proteinuria (albumin and high molecudisturbances of haemodynamics (29), complement ac- lar mass proteins) in contrast to the "minimal lesions" tivation occurs (30), and defects appear in the mem- of glomerular capillaries. brane (gaps, Spiro (31), Stejskal & coworkers (32)), resulting in an unselective proteinuria; larger defects of the membrane, including the basal membrane, even 3.0 Characteristics of Glomerular Protein Permeabillead to an enhanced leakiness for red cells, i.e. miity under Experimental Conditions crohaematuria. In healthy individuals, 80% of eryth3.1 Charge selectivity rocytes excreted in the urine (2 — 4 χ 106/day) are said to be of glomerular origin (33). Statistically, it Multiple experimental techniques have been develseems nearly impossible to detect these physiological oped to obtain an insight into the mechanisms of the Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 10

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barrier function of glomerular capillaries (11). Using polycationic substances (16,40,41), it has been shown that titration (neutralization) of local charges results in an enhanced albumin permeability, which is transient in vivo due to the breakdown of these polycations. Retraction of foot processes of epithelial cells and fusion, which occur in "minimal lesions", were also observed under these conditions. In contrast to in vivo experiments, it is possible in vitro (isolated perfused rat kidney) to neutralize charges step by step permanently using protamine chloride and to induce a dose-related increase of albumin permeability, which almost attains the sieving coefficient of the neutral dextran of similar size (42, 43). In this model it is possible to separate neutralization of lamina rara interna and lamina rara externa from the effect of foot process retraction. The latter is observed only at a higher dose, whereas enhanced albumin permeability increased to a maximum at a lower dosage. Moreover, it was shown in parallel under the influence of protamine that there is no significant change in the size selectivity for neutral macromolecules (dextrans) at ultrastructurally intact podocytes (43, 44).


NaCI perfusion

Fig. 1. Schematic arrangement for reperfusion of rat kidneys after perfusion fixation by glutaraldehyde. NaCl-perfusion was used as a single pass to clean up glomeruli and tubuli between perfusion periods with different protein solutions (45). Pa = mean arterial pressure, Bl, B2, B3 = different protein solutions 0.10CH

When the negatively charged protein cores in the lamina rara interna and lamina rara externa are anchored within the collagen network by glutaraldehyde '·= 0.050fixation of the kidney, the effect of protamine upon < albumin permeability in the reperfused kidney (tech- 0 ü nique see fig. 1) is dramatically reduced (45) (figs. 2, 3). We have learned from these data that dye techniques using polycations expand the charged mesh and produce "electric gaps" by concentrating charges, but only as long as protein cores are flexible and before they have been anchored definitively by glu0.000taraldehyde within the collagen network (45). To4 5 6 7 8 9 gether with Flohr, we showed that the permeability pH of the perfusate for albumin can be reduced under the influence of monocationic cetylpyridinium chloride, possibly by Fig. 2. Sieving coefficient for albumin (ratio of the clearance values of albumin and inulin) in the reperfused rat blockade of individual passage routes (45). In this kidney after perfusion fixation in relation to pH of the case there may be no concentrating and complexing perfusate. Albumin perfusates were prepared in different of charges which concomitantly will produce charge chemical buffers (citrate, phosphate, borate), producing different pH-values to change the surface charge of the gaps. These experiments demonstrate that a selective albumin molecules. At pH values below the isoelectric increase of the permeability of albumin is possible by point, albumin will be positively charged, which enestablishing charge gaps, which can be effected by hances the permeability of glomerular capillaries dramatically. At increasing pH values permeability deconcentrating charges using polycations such as procreases steadily (44, 45). tamine, without a significant change of size selectivity for neutral macromolecules in the lamina densa of the glomerular basement membrane.

I

3.2 Size selectivity Size selectivity can be investigated in the isolated perfused rat kidney, using clearance and micropuncture techniques. The protein permeability of glomeruli in vitro is much more heterogeneous than that in vivo.

If albumin is used as the only protein during perfusion, albumin permeability is some 10 — 20 fold higher than in vivo (2, 46), a process that attains a steady state 10 min after switching from in vivo perfusion with blood to in vitro cell-free perfusion (47) (see tab. 1). The analysis of 98 early proximal micropuncture Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 10

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Schurek et al.: Pathophysiology of glomerular permeability 40

0.020-1

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100

200

300 400 Albumin [mg/l]

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Fig. 3. Sieving coefficient for albumin (ratio of the clearance values of albumin and inulin) in the reperfused kidney after perfusion fixation, in relation to pH of the perfusate. At pH 7.4 and 8.75 addition of protamine (300 mg/l) resulted in a small increase of permeability compared with the intact isolated perfused rat kidney (45).

Fig. 4. Frequency distribution of early proximal albumin concentration (n = 98 micropuncture samples) drawn under pressure control in the isolated perfused rat kidney (48), compared with samples drawn under in vivo conditions (19). The shift to the right is comparable to the shift induced by angiotensin II under in vivo conditions (19).

Tab. 1. Filtration, reabsorption and excretion of albumin under control conditions and in the isolated rat kidney perfused with a mono-component albumin solution (1 — 3, 46, 50).

What are the physiological mechanisms for the repair of these defects? Repair is probably the result of the combined action of macromolecules, clotting factors, fibronectin and cellular elements. As this natural material for the repair of defects is not included during cell-free perfusion, when albumin is the only colloid, the masked defects will be exposed or unmasked, and the albumin permeability will increase within ten minutes, most probably by direct passage through these membrane defects. In accordance with this view, proximal tubules contain a heterogenous pattern of reabsorption vacuoles (lysosomes), which may correspond to the 16% of leaky glomeruli revealed by the micropuncture analysis. Addition of erythrocytes reduces albumin permeability by 50 to 80% (47), and originally we thought that this might be due to the closure of gaps by red cell blockage, as shown under pathological conditions (34, 36, 37). This may account for part of the effect, but a definitive explanation for these phenomena is yet to be established. The remaining increased permeability may be due to smaller gaps, which under in vivo conditions may be sealed by covalently bound plasma proteins or other plasma factors. From these results we derived the hypothesis of "repaired defects" (46), which are thought to exist normally in the healthy kidney. A recenly published analysis shows that orosomucoid (αϊ-acid glycoprotein) is a possible candidate sealing protein (51).

Filtration and reabsorption of albumin Sieving coefficient/ clearance ratio χ 10~3 Reabsorption (%) Excretion (%)

Control in vivo

0.27 ± 0.05

In vitro perfused kidney

4.10 ± 0.30

94

10

6

90

η = 11

η = 105

probes (fig. 4) exhibits an inclined frequency distribution with a peak (50% of all values) in the "normal" albumin concentration range (< 40 mg/l) and 16% of values with albumin concentrations > 300 mg/l (46). This frequency distribution reflects the fact that even under physiological conditions there are gaps or defects within the collagen network of the lamina densa, which may be masked under in vivo conditions during the perfusion of blood. Eisenbach and coworkers (19) demonstrated a similar frequency distribution with a high fraction of leaky glomeruli under the haemodynamically disturbing influence of angiotensin II in vivo. Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 10

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3.3 Haemodynamics If angiotensin II is used in the isolated perfused kidney to generate haemodynamic stress, typical haemodynamic effects occur, such as vasoconstriction and reduction in glomerular filtration rate, but in comparison with the situation in vivo, albumin clearance rates show no increase of albumin permeability (see tab. 2). Thus, if the hypothesis of repaired defects is applied (46) in this experimental setting, it is not possible to unmask more defects by haemodynamic stress than already unmasked during the previous cell free perfusion (48). Tab. 2. The effect of angiotensin II on renal plasma flow, glomerular filtration rate, filtration fraction, and on the clearance quotient Caibumm/Qnuiin in the isolated perfused rat kidney (46, 48, 50). Pa 120mmHg Renal plasma (ml/min · g)

flow

Control

Angiotensin II (0-60 min)

33.7 ±2.1

21.2 ±1.0

Glomerular filtration rate (ml/min · g)

1.24 ± 0.06

0.93 ± 0.03

Filtration fraction

3.40 ± 0.3

4.6 ± 0.3

^albumin

5.1 ± 0.5

4.1 ± 0 . 5

n =8

n = 16

(

\(\ — "$\

^inulin

The definitive characteristics of the glomerular permeability for macromolecules may be a combined effect of glomerular porosity (size selectivity), charge pattern and repair mechanisms, which act to seal naturally existing membrane defects with the aid of components provided continually by the blood. A monocomponent perfusion with albumin alone is able to withdraw these components and thus unmask these naturally existing defects. Therefore it is not surprising that the so-called functional proteinuria, which can be demonstrated under defined physical stress conditions even in man, is an unselective proteinuria. It can be demonstrated under the influence of physical stress or vasoconstrictors, under the influence of fever (49), after unilateral nephrectomy (25) and in patients suffering from essential hypertension. It may in part be due to the fact that masked defects may be unmasked under the influence of haemodynamic disturbances.

Acknowledgement This work was supported for 10 years by grant Schu 343 and partly by grant SFB 146, both from the Deutsche Forschungsgemeinschaft. We gratefully acknowledge the support, encouragement and discussions of Enrico Reale and Karlwilhelm Kühn and the cooperation of Jeanette Alt. Parts of the Thesis of Hermann Thole, Horst Pagel, Harald Flohr and Mathias Zeh are included in the material. Parts of this material have been published in 1. c. (50).

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Schurek et al.: Pathophysiology of glomerular permeability 18. Pessina, A. C. & Peart, W. S. (1972) Renin induced proteinuria and the effects of adrenalectomy. I. Hemodynamic changes in relation to function. Proc. R. Soc. Lond. B 180, 43-60. 19. Eisenbach, G. M., van Liew, J. B. & Boylan, J. W. (1975) Effect of angiotensin on the filtration of protein in the rat kidney. A micropuncture study. Kidney Int. 8, 80 — 87. 20. Hulme, B. & Pessina, A. C. (1975) Influence on renin and angiotensin II on macromolecular glomerular permeability. VI. Int. Congr. Nephrol. Symposium, Abstracts pp. 71 —72. 21. Blantz, R. C., Können, K. S. & Tucker, B. J. (1976) Angiotensin II effects upon the glomerular microcirculation and ultrafiltration of the rat. J. Clin. Invest. 57, 419-434. 22. Carrie, B. J., Hilbermann, M., Schroeder, J. S. & Myers, B. D. (1980) Albuminuria and the permselective properties of the glomerulus in cardiac failure. Kidney Int. 77, 507 — 517. 23. Göbel, U, Klenke, K. H., Günther, K. H. & Natusch, R. (1984) Auftreten und Verhalten der Proteinurie bei Herzinsuffizienz. Dtsch. Gesundh. Wesen 39, 653-655. 24. Feld, L., van Liew, J. B., Galaske, R. G. & Boylan, J. W. (1977) Selectivity of renal injury and proteinuria in the spontaneously hypertensive rat. Kidney Int. 72, 332 — 343. 25. Oberle, G., Neumann, H. P. H., Schollmeyer, P., Boesken, W. H. & Stahl, R. A. K. (1985) Mild proteinuria in patients with unilateral kidney. Klin. Wochenschr. 63, 1048-1051. 26. Mogensen, C. E. (1987) Microalbuminuria as a predictor of clinical diabetic nephropathy. Kidney Int. 37, 673 — 689. 27. Winetz, J. A., Robertson, C. R., Golbeth, H. V., Carrie, B. J., Salyer, W. R. & Myers, B. D. (1981) The nature of the glomerular injury in minimal change and focal sclerosing glomerulopathies. Am. J. Kidney Dis. 7, 91—98. 28. Carrie, B. J., Salyer, W. R. & Myers, B. D. (1981) Minimal change nephropathy: An electrochemical disorder of the glomerular membrane. Am. J. Med. 70, 262 — 268. 29. Stahl, R. A. K. (1986) Die Bedeutung von Eicosanoiden bei glomerulären Erkrankungen. Klin. Wochenschr. 64, 813-823. 30. Bitter-Suermann, D. (1983) Das Komplementsystem: Funktion und klinische Bedeutung. Dtsch. Ärzteblatt 80, 33-48. 31. Spiro, D. (1959) The structural basis of proteinuria in man. Am. J. Pathol. 35, 47-73. 32. Stejskal, J., Pirani, C. L., Okada, M., Mandelanakis, N. & Pollak, V. E. (1973) Discontinuities (gaps) of the glomerular capillary wall and basement membrane in renal diseases. Lab. Invest. 28, 149-169. 33. Fairley, K. F. & Birch, D. F. (1982) Hematuria: A simple method for identifying glomerular bleeding. Kidney Int. 27, 105-108. 34. Bohle, ., von Giese, H. & Mikeler, E. (1984) Zur Pathogenese der Makrohämaturie bei IgA-Nephritis. Pathologe 5,322-325. 35. Sato, K. & Ullrich, K. (1975) Serum-induced inhibition of isotonic fluid absorption by the kidney proximal tubule. III. Further evidence that complement mediated cell lysis is involved. Biochim. Biophys. Acta 477, 144—154. 36. Lin, J. T., Wada, H., Maeda, H., Hattori, M., Tanaka, H., Uenoyama, F., Suehiro, A., Noguchi, K. & Nagai, K. (1983) Mechanism of hematuria in glomerular disease. Nephron35, 68-72.

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37. Bohle, ., von Giese, H., Mikeler, E. & Rassweüer, J. (1985) Morphologic contribution on gross hematuria in mild mesangioproliferative glomerulonephritis without crescents. Klin. Wochenschr. 63, 371-378. 38. Hofmann, W., Schmidt, D., Guder, W. G. & Edel, H. H. (1991) Differentiation of hematuria by quantitative determination of urinary marker proteins. Klin. Wochenschr. 69, 68-75. 39. Shemes, O., Ross, J. C., Deen, W. M., Grant, G. W. & Myers, B. D. (1986) Nature of the glomerular capillary injury in human membranous glomerulopathy. J. Clin. Invest. 77, 868-877. 40. Hunsicker, C. G., Shearer, T. P., Shaffer, S. J. (1981) Acute reversible proteinuria induced by infusion of the polycation hexadimethrine. Kidney Int. 20, 7 — 17. 4L Seiler, M. W., Rennke, H. G., Venkatachalam, . ., Cotran, R. S. (1977) Pathogenesis of polycation induced alterations ("fusion") of glomerular epithelium. Lab. Invest. 36, 46-61. 42. Assel, E., Neumann, K. H., Schurek, H. J., Sonnenburg, C. & Stolte, H. (1984) Glomerular albumin leakage and morphology after neutralization of polyanions. I. Albumin clearance and sieving coefficient in the isolated perfused rat kidney. Renal Physiology 7, 357-364. 43. Sonnenburg-Hatzopoulos, C., Assel, E., Schurek, H. J. & Stolte, H. (1984) Glomerular albumin leakage and morphology after neutralization of polyanions. II Discrepancy of protamin induced albuminuria and fine structure of the glomerular filtration barrier. J. Submicrosc. Cytol. 26, 741-751. 44. Zeh, M. (1992) Untersuchungen zur Permselektivität für Makromoleküle an der fixierten, reperfundierten Rattenniere. Thesis. Medizinische Hochschule Hannover. 45. Flohr, H. (1991) Die Bedeutung der negativen Ladungen in der glomerulären Basalmembran für die Proteinurie. Untersuchungen an dem neu etablierten Modell der reperfundierten, isolierten und anatomisch fixierten Rattenniere. Thesis, Med. Hochschule Hannover. 46. Schurek, H. J., Pagel, H., Thole, H., Neumann, K. H., Alt, J. M., Bahlmann, J. & Stolte, H. (1986) Die "Repaired Defect" Hypothese der glomerulären Kapillarwand. Untersuchungen an der isoliert perfundierten Niere. Abstract. Nieren u. Hochdruckkrankheiten 75, 368. 47. Pagel, H., Schurek, H. J. & Stolte, H. (1985) Untersuchungen zur funktionellen Filtrationsbarriere für Albumin an der isoliert perfundierten Rattenniere. Herbsttagung der Dtsch. Physiol. Gesellschaft, Berlin. 48. Thole, H. (1988) Zum Mechanismus der Angiotensin II induzierten Proteinurie. Thesis. Medizinische Hochschule Hannover. 49. Boesken, W. H., Marnier, ., Neumann, H. & Engelhardt, R. (1983) Gibt es die "Febrile Proteinurie"? Klin. Wochenschr. 61, 917-922. 50. Schurek, H. J. (1987) Physiologische und Pathophysiologische Grundlagen der glomerulären Permeabilität für Plasmaproteine. Verh. Dtsch. Ges. Inn. Med. 93, 466-472. 51. Haraldsson, B. S., Johnsson, E. K. A. & Rippe, B. (1992) Glomerular permselectivity is dependent on adequate serum concentrations of orosomucoid. Kidney Int. 41, 310—316. Prof. Dr. H.-J. Schurek St. Bonifatius-Hospital Abt. Nephrologie POB 2040 W-4450 Lingen/Ems Bundesrepublik Deutschland

Schleicher and Olgemöller: Glomerular changes in diabetes mellitus

635


Glomerular Changes in Diabetes Mellitus By E. D. Schleicher and B. Olgemöller Institut für Diabetesforschung München (Received February 12/July 13, 1992)

Summary: Ultrastructural, immunohistochemical and biochemical studies have improved our knowledge on the events occurring during the development of diabetic late complications. Immunohistochemical investigations of diabetic kidneys, using antibodies against various components of the extracellular matrix, showed increased collagen type IV (oti,a2-chain) deposition in the mesangial matrix, and a decrease of heparan sulphate proteoglycan in the mesangial matrix and glomerular basement membrane. Changes in matrix components seem to be the underlying cause of the alterations in renal function, as reflected by albuminuria and proteinuria. The occurrence of collagen type III in late diffuse glomerulosclerosis has been interpreted as an irreversible change in glomerular structure. The extent of alteration of the extracellular matrix correlates to a certain extent with the severity of nephropathy of the individual subject. The studies performed to date support the hypothesis that hyperglycaemia, whatever its origin, is the primary cause of diabetic late complications, although the pathobiochemical mechanisms are not yet fully understood. Increased intra- and extracellular levels of glucose and its derivatives are thought to contribute to diabetic tissue dysfunction. Three pathobiochemical theories are favoured in the current discussion: i) the polyol pathway ii) non-enzymatic glycation of proteins iii) direct influence of hyperglycaemia on the synthesis of matrix components. The evidence for the participation of the polyol pathway in the pathogenesis of diabetic nephropathy comes mainly from animal data using aldose reductase inhibitors, but only limited data are available for humans, so that the significance of this pathomechanism cannot yet be determined. The formation of non-enzymatically glycated proteins in blood and tissue and the subsequent rearrangements in a slow series of chemical reactions lead to "Advanced Glycation End products". These "AGE-products" may directly stimulate kidney cells or macrophages to secrete cytokines or growth factors which induce matrix alterations in an auto/paracrine manner. In vitro studies with cultured kidney cells show that the dysregulation of the biosynthesis of collagen type IV and heparan sulphate proteoglycan can also be caused directly by increased glucose concentrations.

Introduction

~ , . , , . _ Diabetic nephropathy is now Althe mostA common cause , j. TT *· · j off end-stage renal disease. Heavy proteinuria and . i i r *· u· «. r t progressive loss of glomerular function ultimately re,. . . ~ . suit m renal insufficiency. Although Kimmelstiel & Wilson (1) described diabetic glomerulosclerosis more than 50 years ago, progress in elucidating the pathobiochemistry of this form of diabetic microangiopathy has been slow. This may be Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 10

in part due to the fact that the molecular organisation ~ , ~, . . . ,. of the filtration unit in mammalian kidneys was not „ , , . , , ,^ we U understood. As reviewed recently (2), progress , , , . , .. , , has been made in characterizing ö the ultrastructure and the composition of the glomerulum both by electron microscopic and biochemical methods. These investigations have led to an improved understanding of the structure of the glomerulum on a molecular basis.

636

Schleicher and Olgem ller: Glomerular changes in diabetes mellitus

In addition, more recent studies have improved our knowledge of the structure-function relationship of the glomerular filtration unit.

Component

Structure

Function

Structure and Function of Normal Glomerula

Collagen type IV

Tripel helix with segments; 5 different chains with ca. 1700 aminoacids are known.

Mechanical scaffold Size-selective filter

oc,a2-chain

Occurs in mesangial matrix, Bowman's capsule and glomerular basement membrane.

a304-chain

Only in the glomerular basement membrane

Laminin

Three different polypeptide Cell adhesion chains A,, B,, B2, Mt 800000

Heparan sulphate proteoglycan

Core protein Μτ 470000 Three heparan sulphate side chains.

Charge-selective filter

Entactin

One polypeptide chain Mr 140000

Associated with laminin

The glomerular basement membrane represents the size- and charge-selective barrier of the glomerulum. It is composed of a backbone of type IV collagen, which makes up the lamina densa, flanked on either side by layers with different composition, the laminae rarae interna and externa. The latter contains heparan sulphate proteoglycan and attachment proteins like laminin, entactin and other glycoproteins. A detailed description of the components of the glomerular extracellular matrix and their molecular properties is given in table 1. The proteoglycan-containing layer provides a viscous, negatively charged screen in front of the lamina densa. In this location the heparan sulphate proteoglycan seems to be ideally suited to play a major role in the filtration process. Based on their known properties, the proteoglycans would be expected to retard filtration of macromolecules by both electrostatic repulsion and steric hindrance, thereby essentially contributing to the charge-selective barrier of the glomerular basement membrane. In vivo studies indicate that specific digestion of the heparan sulphate side chain leads to penetration of anionic proteins such as albumin and ferritin into and through the glomerular basement membrane. Therefore, it was concluded that the heparan sulphate proteoglycan is essential to the maintenance of the integrity of the glomerular permeability barrier to macromolecules (3 — 5). Although there has been considerable debate on the diversity of renal heparan sulphate proteoglycans, it is now generally accepted that the proteoglycan associated with glomerular basement membrane consists of 3 heparan sulphate side chains covalently attached to a large monomeric polypeptide chain, the structure of which has been deduced from cDNA obtained from different sources by three independent groups (6 — 8). Elegant work of van den Born et al. (9) has unequivocally shown that the heparan sulphate side chains form the anionic barrier of the glomerular filtration unit, since intravenous injection of monoclonal antibodies directed against the heparan sulphate moiety resulted in albuminuria in rats. This antibody-induced albuminuria was reversible and was accompanied by transient binding of the injected antibody to the glomerular basement membrane. The mesangial matrix, although developmentally and morphologically distinct from the glomerular basement membrane, is composed of essentially the same components i.e. collagen IV, laminin and heparan

Tab. 1. Biochemical data of glomerular basement membrane components

sulphate proteoglycan. Furthermore, fibronectin, entactin, collagen V and VI are found in this glomerular compartment. Recently, it was recognized that the collagen IV chains were differently distributed within the glomerulum. Using immunohistochemical methods, the αΐ5α2chains were primarily detected in the mesangial matrix, whereas the oc3,a4-chains were exclusively found in the glomerular basement membrane (10) (tab. 1). Structural and Functional Changes in Diabetic Glomerula

Thickening of glomerular basement membrane and expansion of the mesangial matrix are hallmarks of the morphological changes observed in human diabetic nephropathy. Therefore, numerous studies using ultrastructural or immunohistochemical methods have addressed the increased deposition of extracellular matrix components in diabetes mellitus. The first major change after onset of diabetes is the increased volume of the whole kidney (11) and the glomerula (12). These hypertrophical glomerula have normal structural composition. After a few years the amount of matrix of the peripheral glomerular basement membrane and the mesangial matrix is increased. Immunohistochemical studies with monospecific antibodies against the various basement membrane components have revealed changes in the extracellular matrix during the development of diabetic nephropathy (10,13). The essential findings are summarized in table 2 and will be described briefly. Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 10

Schleicher and Olgem ller: Glomerular changes in diabetes mellitus Tab. 2. Composition of the glomerular matrix in different stages of diabetes mellitus (10, 13)

Collagen IV (^\CL2) Collagen IV (α3α4) Laminin Heparan sulphate proteoglycan a) Fibronectin Collagen V Collagen III Collagen I

Diffuse glomerular sclerosis

Nodular glomerular sclerosis

Early

Late

Early

ί

ΐί

ΐ ί

1Τ i-

I ΐ i1

I

ί

4 1

Late

4 1 1

637

It appears that collagen IV, laminin, fibronectin and collagen V increase during the development of diabetic nephropathy, whereas heparan sulphate proteoglycan decreases very early. Collagen III is not found in normal glomeruli but occurs in late diffuse glomerulosclerosis and increases further during the development of nodular glomerulosclerosis. The occurrence of collagen III in the mesangium may reflect the irreversibility of the process.

l i

Nerlich et al. (13) compared their immunohistochemical findings with the available clinical data (serum Τ Ι creatinine concentration, creatinine clearance and ΐ ί Τ ί Τ ί Τ proteinuria) for the evalution of renal function. In ΐ (+) (+) — — — — two patients without reports of impaired filter function, a slight reduction in the glomerular heparan Ι = increase, | = decrease, —» = no change, + = present, sulphate proteoglycan was found. In the other cases — = not detectable a) The preparation and characterization of the antigen and the they found consistently reduced proteoglycan content antiserum is described in 1. c. (37). and impaired filter function. Independent determination of glomerular heparan sulphate proteoglycan from normal and diabetic subjects confirmed the decrease of heparan sulphate proteoglycan in diabetic In diabetic kidneys with slight lesions (early diffuse glomeruli. These measurements were performed with glomerulosclerosis) only a minor increase in all base- an immunoassay (14) and by biochemical methods ment membrane components was found, except for (15).

τ

τ

heparan sulphate proteoglycan, which was present in the glomerular basement membrane and thickened mesangial matrix, although with decreased staining intensity. More pronounced diffuse glomerulosclerosis showed a further increase in basement membrane components, and especially in collagen IV al5a2-chains in the mesangial matrix. Collagen V also showed an increased staining pattern. However, heparan sulphate proteoglycan was entirely absent from the enlarged matrix. Minor amounts of heparan sulphate proteoglycan could only be observed in the periphery of the glomeruli. The staining of collagen IV a3,a4-chain showed a similar distribution with intense staining of the thickened glomerular basement membrane. The composition of small nodular matrix enlargement resembles that of diffuse glomerulosclerotic lesions. Again, a marked increase of basement membrane collagen IV and collagen V was found, as well as small increases of laminin and fibronectin, whereas heparan sulphate proteoglycan was not detectable. In contrast, pronounced nodular lesions exhibited a strong decrease of collagen IV a1,a2-chain, laminin, and fibronectin. These components were only present in the periphery of the noduli. In the hypocellular central areas of the noduli, only collagen V could be found. Furthermore, peripheral areas of these noduli were also positive for collagen III. Collagen I was never detectable in diffuse or in nodular lesions. This is in marked contrast to reparative processes, since during woundhealing collagen I is formed. Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30, 1992 / No. 10

Pathobiochemical Events Causing Diabetic Nephropathy Although still controversial, most data indicate that a liability for the development of diabetic nephropathy is linked to hyperglycaemia. Animal studies have provided fairly strong support for a direct link between hyperglycaemia and glomerular changes (16). Several pathomechanisms have been suggested, linking increased glucose concentration with changes in diabetic kidneys. Three biochemical pathways which describe the most promising pathomechanisms for the development of diabetic glomerulopathy are discussed below. Non-Enzymatic Glycation and "Advanced Glycation End Product" Formation Through extensive studies on the formation and structure of glycated haemoglobin, the chemistry of nonenzymatic glycation has been well established. As indicated by the name, glucose reacts without enzymatic catalysis unspecifically with free amino groups of a given protein. Non-enzymatic glycation proceeds slowly with time. In contrast, the enzymatic glycosylation of proteins takes place at defined serine, threonine or asparagine residues in the Golgi apparatus with the aid of a complex enzymatic machinery. The amount of glucose bound to protein by non-enzymatic glycation is proportional to the glucose concentration

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Schleicher and Olgem ller: Glomerular changes in diabetes mellitus

and depends also on the half-life of the protein in question (17). Therefore, investigations of a variety of tissues from diabetic and non-diabetic subjects showed that the level of non-enzymatically bound glucose was increased in those tissues known as the preferred targets of diabetic late complications, i. e. arterial blood vessels, glomerular basement membrane, connective tissues and peripheral nerves. Furthermore, the amount of non-enzymatically bound glucose in these tissues seemed to be related to the extent and severity of the patients' late complications (18). Glycation of proteins blocks free amino groups of lysine residues, which may therefore no longer be available for fulfilling their normal physiological functions. Furthermore, glycation may also cause unphysiological cross-linking of proteins as indicated by changes in the physical properties of glycated collagen, like decreased solubility and greater thermal resistance (19). However, more recent findings show that the development of diabetic late complications may also be explained by the formation of "advanced glycation end products" recently identified by Cerami's group (20, 21). The "advanced glycation endproducts" arise first of all in proteins with long life times and result from the reaction of glycated lysyl residues with free amino groups, forming unphysiological cross-links. In addition, these "advanced glycation end-products" lead to secretion of cytokines when bound to a specific "advanced glycation endproduct receptor" recently identified on macrophages (22). These cytokines may cause increased synthesis of extracellular matrix components and may influence vascular permeability. Recently, it has been shown that "advanced glycation end-products" act on mesangial cells via platelet-derived growth factor causing the cells to synthesize increased amounts of collagen IV, laminin and heparan sulphate proteoglycan (23). The formation of the above mentioned abnormal unphysiological cross-links and the release of cytokines (24) can be prevented by aminoguanidine, a compound that inhibits the formation of advanced glycation end products. Thus, diabetic rats fed with aminoguanidine for 7 months showed significantly less pathological cross-links of glomerular basement membrane collagen when compared with untreated animals (25). Furthermore, the thickening of the glomerular basement membrane was prevented by administration of aminoguanidine. Although the results of this pharmacological intervention look quite promising, more studies are needed to determine whether aminoguanidine may also prevent the development of nephropathy with subsequent endstage renal failure.

Polyol Pathway and Diabetic Nephropathy An increase in intracellular sorbitol concentration following hyperglycaemia has been implicated in the development of diabetic late complications by various researchers, and has been summarized previously by Greene (26). It has been shown that chronic hyperglycaemia leads to sorbitol accumulation in different tissue, such as peripheral neurons (26), lens and renal tubuli (27). The initial hypothesis that sorbitol accumulation causes effects which might damage tissues is unlikely to operate in kidney (28). The inositol depletion theory suggested by Greene and coworkers explains tissue damages by impairment of myo-inositol uptake leading to depletion of phosphatidylinositides (26). Although the cellular inositol uptake is competitively inhibited by D-glucose (29) our recent studies show that cells may counterregulate impaired inositol uptake (unpublished). Furthermore, it is not generally agreed whether the increase in intracellular sorbitol is the cause of the impaired function of the affected tissues in diabetes. After treatment of diabetic rats for six months with the aldose reductase inhibitor, tolrestat, only a slight reduction in the urinary albumin excretion rate could be demonstrated, indicating that other pathomechanisms are operating in diabetic glomerulopathy (30).

Effect of Glucose on the Synthesis of Basement Membrane Components Several authors have studied the expression of basement membrane components in diabetic animals. Ledbetter and associates (31) found that the levels of mRNA for the αϊ-chain of collagen IV in kidneys of diabetic rats were increased when assayed by northern blot analysis. Accordingly, Lorenzi and coworkers (32) found elevated levels of mRNA for fibronectin and collagen IV, and unchanged levels of mRNA for collagen I and c-myc in cultured endothelial cells grown in the presence of elevated glucose concentrations. Prolonged incubations of mesangial cells in high glucose media caused an increased production of collagen IV, laminin and fibronectin (33, 34). All these results are in agreement with increased collagen production in the diabetic state. We have investigated whether heparan sulphate proteoglycan synthesis is influenced by glucose in mesangial cells (35). The results showed that increasing glucose concentrations decreased heparan sulphate proteoglycan synthesis within two days. Under the same conditions, elevated glucose concentrations had no effect on the heparan sulphate proteoglycan content of endothelial cells.



639

Recent findings suggest that elevated glucose concentrations directly influence the biosynthesis of extraThe recent findings corroborate and supplement the cellular matrix components. Scheme 1 summarizes the pathogenetic hypothesis of Rohrbach et al. (36). Acevents which may lead to diabetic glomerulosclerosis. cording to their biochemical findings, they postulated that a reduction in the glomerular heparan sulphate Hyperglycaemia proteoglycan content may lead to an increased synI Loss of heparan sulphate proteoglycan thesis of basement membrane components, resulting Increased biosynthesis of collagen type IV and fibronectin in a progessively thickened basement membrane. A (mesangial expansion and basement membrane thickening) recurrent diabetic metabolic injury of the glomerular I Impaired glomerular filtration barrier basement membrane may thus lead to the multilam(albuminuria) inated deposition of basement membrane material, I Synthesis of unphysiological matrix components which lacks, however, heparan sulphate proteoglycan. (sclerosis) In addition to this hypothesis it was demonstrated Reduction of filtration surface that the late stage of diabetic glomerulopathy is charRenal failure acterized by occurrence of unphysiological matrix material with progressive sclerosis of the glomerulum. Scheme 1. Pathogenesis of diabetic nephropathy The occurrence of collagen III in the mesangium may reflect irreversibility. The dysregulation of the com- Acknowledgement ponents of the extracellular matrix is well documented The experimental work was supported by the Deutsche Forin cell culture studies, animal models and in man. schungsgemeinschaft (Schi 239/1-3 and Olge 48/1-3). Conclusions

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10. Kim, Y, Kleppel, M., Butkowski, R., Mauer, M., Wieslander, J. & Michael, A. (1991) Differential Expression of Basement Membrane Collagen Chains in Diabetic Nephropathy. Amer. J. Pathol. 138, 413-420. 11. Steffens, M. W, Osterby, R., Chavers, B. & Mauer, M. S. (1989) Mesangial Expansion as a Central Mechanism for Loss of Kidney Function in Diabetic Patients. Diabetes 38, 1077-1081. 12. Osterby, R. & Gunderson, H. J. G. (1975) Glomerular size and structure in diabetes mellitus: early abnormalities. Diabetologia 11,225-259. 13. Nerlich, A. & Schleicher, E. (1991) Immunohistochemical localization of extracellular matrix components in human diabetic glomerular lesions. Am. J. Pathol. 139, 889-899. 14. Shimomura, H. & Spiro, R. G. (1987) Studies on macromolecular components of human glomerular basement membrane and alterations in diabetes: decreased levels of heparan sulfate proteoglycan. Diabetes 36, 374—381. 15. Schleicher, E. & Wieland, O. H. (1984) Changes of human glomerular basement membrane in diabetes mellitus. J. Clin. Chem. Clin. Biochem. 22, 223-227. 16. Kern, T. S. & Engerman, T. L. (1990) Arrest of glomerulopathy in diabetic dogs by improved diabetic control. Diabetologia 21, 178-183. 17. Schleicher, E. D. & Wieland, O. H. (1986) Kinetic analysis of glycation as a tool for assessing the half-life of proteins. Biochim. Biophys. Acta 884, 199-205. 18. Vogt, B. W., Schleicher, E. D. & Wieland, O. H. (1982) aminolysine bound glucose in human tissues obtained at autopsy: increase in diabetes mellitus. Diabetes 31, 1123 — 1127. 19. Bailey, J. A. & Kent, M. J. C. (1989) Non-enzymatic glycosylation of fibrous and basement membrane collagens. In: The Maillard Reaction in Aging, Diabetes and Nutrition (Baynes, J. & Monnier, V., eds.) Alan R. Liss, Inc. New York, 109-122.

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20. Brownlee, M., Cerami, A. & Vlasssara, H. (1988) Advanced glucosylation end products in tissue and the biochemical basis of diabetic complications. N. Engl. J. Med. 318, 1315-1321. 21. Ledl, F. & Schleicher, E. (1990) New Aspects of the Maillard reaction in foods and in the human body. Angew. Chem. Int. Ed. Engl. 29, 565-594. 22. Vlassara, H., Brownlee, M. & Cerami, A. (1986) Novel macrophage receptor for glucose-modified proteins is distinct from previously described scavenger receptors. J. Exp. Med. 164: 1301-1309. 23. Doi, T., Vlassara, H., Kirstein, M., Yamada, Y., Striker, G. E. & Striker, L. J. (1992) Receptor-specific increase in extracellular matrix production in mouse mesangial cells by advanced glycosylation end products is mediated via platelet-derived growth factor. Proc. Natl. Acad. Sei. USA 89, 2873-2877. 24. Vlassara, H., Brownlee, M., Manogue, K. R., Dinarello, C. A. & Pasagian, A. (1988) Cachectin/TNF and IL-1 induced by glucose-modified proteins: role in normal tissue remodeling. Science 240, 1546-1548. 25. Brownlee, M., Vlassara, H., Kooney, T., Ulrich, P. & Cerami, A. (1986) Aminoguanidine prevents diabetes-induced arterial wall protein cross-linking. Science 232,1629 — 1632. 26. Greene, D. (1988) The pathogenesis and its prevention of diabetic neuropathy and nephropathy. Metabol. 37, Suppl. 1, 25-29. 27. Schmolke, M., Schleicher, E. D. & Guder, W. G. (1992) Renal sorbitol, myoinositol and glycerophosphorylcholine in streptozotocin-diabetic rats. Eur. J. Clin. Chem. Clin. Biochem. 30, 607-614. 28. Larkins, R. G. & Dunlop, M. E. (1992) The link between hyperglycaemia and diabetic nephropathy. Diabetologia 35, 499-504. 29. Olgemöller, B., Schwaabe, S., Schleicher, E. D. & Gerbitz, K. D. (1990) Competitive inhibition by glucose of myoinositol incorporation into cultured porcine mesangial cells. Biochim. Biophys. Acta 7052, 47-52.

30. Me Caleb, M. L., Me Kean, M. L., Hohman, T. C., Laver, N. & Robinson, W. G. (1991) Intervention with aldose reductase inhibitor, tolrestat, in renal and retinal lesions of streptozotocin diabetic rats. Diabetologia 34, 659 — 701. 31. Ledbetter, S. R., Copeland, E. J., Noonan, D., Vogeli, G. & Hassel, J. R. (1990) Altered steady-state in mRNA levels of basement membrane proteins in diabetic mouse kidneys and thromboxane synthase inhibition. Diabetes 39, 196— 203. 32. Cagliero, E., Maiello, M., Boeri, D., Roy, S. & Lorenzi, M. (1988) Increased expression of basement membrane components in human endothelial cells cultured in high glucose. J. Clin. Invest. 82, 735-738. 33. Ayo, S. H., Radnik, R. A., Glass, II W. F., Garoni, J. A., Rampt, E. R., Appling, D. R. & Kreisberg, J. I. (1990) Increased extracellular matrix synthesis and mRNA in mesangial cells grown in high-glucose medium. Amer. J. Physiol. 260, F185-F191. 34. Haneda, M., Kikkawa, R., Horide, N., Togawa, M., Koya, D., Kajiwara, N., Ooshima, A. & Shigeta, Y. (1991) Glucose enhances type IV collagen production in cultured rat glomerular mesangial cells. Diabetologia 34, 198 — 200. 35. Olgemöller, B., Schwaabe, S., Gerbitz, K. D. & Schleicher, E. D. (1992) Elevated glucose decreases the content of a basement membrane associated proteoglycan in proliferating mesangial cells. Diabetologia 35, 183 — 186. 36. Rohrbach, D. H., Hassell, J. R., Kleinman, H. K. & Martin, G. R. (1982) Alterations in the basement membrane (heparan sulphate) proteoglycan in diabetic mice. Diabetes 31, 185-188. 37. Olgemöller, B., Schleicher, E., Nerlich, ., Wagner, E. & Gerbitz, K. D. (1990) Isolation, characterization and immunological determination of basement membrane-associated heparan sulfate proteoglycan. Biol. Chem. HoppeSeyler370, 1321-1229. Dr. E. Schleicher Institut für Diabetesforschung Kölner Platz l W-8000 München 40 Bundesrepublik Deutschland


Schaefer et al.: Proteinases in glomerulosclerosis

641


Role of Glomerular Proteinases in the Evolution of Glomerulosclerosis*) By R. M. Schaefer1, L. Paczek2, S. Huang\ M. Teschner1, Liliana Schaefer1 and A. Heidland1 1 2 3

Department of Nephrology, University of W rzburg, W rzburg, Germany Warsaw Medical School, Transplantation Institute, Warsaw, Poland Department of Medicine, Case Western Reserve University, Cleveland, OH, USA

(Received February 24/July 2, 1992)

Summary: Recent studies suggest that proteolytic enzymes located within the glomerulus are involved in the degradation of extracellular matrix components. In the present investigation glomerular proteinase activities were followed in a variety of non-immune-mediated renal diseases as well as during different dietary manipulations. Azocaseinolysis was significantly reduced in the obese Zucker rat compared with lean littermates (pH 5.4:8.9 ± 0.4 vs 11.4 ± 0.7; pH 7.4: 5.8 ± 0.7 vs 9.3 ± 0.6 arb. U/mg protein). When the glomerular proteolytic capacity was measured in old rats, again a significant decline in proteolysis was observed (pH 5.4: 9.8 ± 0.8 vs 17.7 ± 0.8; pH 7.4: 6.4 ± 0.7 vs 11.7 ± 0.5 arb. U/mg protein). In Goldblatt hypertensive rats the undipped kidney, which is exposed to high blood pressure, revealed lower glomerular azocaseinolytic activity compared with the contralateral clipped kidney (pH 5.4: 8.1 ± 0.4 vs 12.9 ± 0.5 arb. U/mg protein). In parallel, the cathepsin B content was also diminished in glomeruli from kidneys exposed to hypertension. When proteinases were followed in glomeruli from intact kidneys of rats fed protein-modified diets (fraction of casein 0.05, 0.20 or 0.60) a significant fall in the activities of cysteine proteinases, e. g. cathepsin B (casein 0.05: 1,498 + 110 vs casein 0.60: 914 ± 84 μΐΐ/μζ DNA), as well as metalloproteinases, e.g. collagenase (casein 0.05: 233 ± 14 vs casein 0.60: 137 + 11 μΐΐ/^ DNA), occurred. These data indicate that in both early and late stages of glomerulosclerosis, proteolytic activities within the glomerulus tend to be reduced, which could allow extracellular matrix accumulation. Moreover, changes in dietary protein intake resulted in profound alterations of glomerular proteinases. A finding that might explain the beneficial effects of low-protein diets in chronic renal failure.

Excessive deposition of extracellular matrix represents a central feature of the pathomorphology of the glomerulus, both in animals as well as humans. Such accumulation seems to be one of the early events in the evolution of glomerulosclerosis which eventually will

Organization: Deutsche Forschungsgemeinschaft ') Enzymes Cathepsin B EC 3.4.22.1 Cathepsin H EC 3.4.22.16 Cathepsin L EC 3.4.22.15 Collagenase EC 3.4.4.19 Gelatinase EC 3.4.4.19 Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30, 1992 / No. 10

lead to distortion of the glomerular architecture and to a progressive decline in glomerular filtration (1). Extracellular matrix in the glomerulus consists of proteoglycans, glycoproteins, and collagens (2). In general, accumulation of this matrix occurs either by enhanced synthesis or by reduced degradation of these elements, Two classes of proteolytic enzymes1), cysteine proteinases (cathepsins B, H, and L) and metalloproteinases (collagenase and gelatinase), are localized within the glomerulus. The cysteine proteinases are a diverse group of lysosomal enzymes with a broad spectrum r u. Γ Λ ττ *· - .u -Λ°f substrates and a pH optimum in the acidic range (3). Among the best characterized of these enzymes

642

are the cathepsins B, H, and L. Their ability to degrade intact glomerular basement membrane in vitro has been demonstrated by Davies and coworkers (4 — 6) and more recently by Baricos et al. (7). The presence of these enzymes has been established by measurements of enzyme activities (8), by immunohistochemistry (9), and by fluorescence microscopy (8). Lovett and coworkers (10, 11) were the first to describe an enzyme with gelatinase activity which was found in the medium of cultured rat mesangial cells. This enzyme was found to be a metalloproteinase which is secreted by both rat and human (12) mesangial cells. As with other matrix-degrading metalloproteinases, this enzyme is secreted as an inactive zymogen, which is activated by limited proteolysis (10). Maximal activity is achieved at neutral pH. Gelatin, glomerular basement membrane, and soluble type IV collagen are degraded, but not interstitial collagens, proteoglycans, or fibronectin (12) What is more, mesangial cells also secrete an inhibitor of gelatinase with the characteristics of a tissue inhibitor of metalloproteinases (TIMP-1) (13). There is further evidence that rat glomeruli contain more metalloproteinases, such as stromelysin and typ IV collagenase. For these reasons, it is not surprising that incubation of isolated glomeruli with intact glomerular basement membrane at pH 7.4 resulted in considerable glomerular basement membrane breakdown, which could be inhibited by known metalloproteinase inhibitors, but not by inhibitors of serine or cysteine proteinases (14). In the present study intraglomerular proteolytic activities were investigated in a variety of animal models for non-immune-mediated glomerulosclerosis, such as the obese Zucker rat (15), the aging kidney (16), and the kidney in severe hypertension (17), to determine whether the process of glomerulosclerosis was associated with altered proteinase activities. In addition, the effect of dietary protein on glomerular proteinase activity was evaluated (18).

Methods Animals Groups of three animals were housed in single cages at constant humidity and temperature with a 12 h light/dark cycle. All animal strains used in the different experiments were purchased from Savo-Ivanovas, Kisslegg, Germany and handled as follows: 1) The obese and lean Zucker rats were fed a standard rat chow (Altromin, Lippe, Germany). These animals were sacrificed at 16 weeks of age. 2) For the experiments addressing the aging kidney, 27-monthsold male Wistar rats (weighing 440 g) were compared with 6-months-old rats of the same weight.

Schaefer et al.: Proteinases in glomerulosclerosis 3) In the model of two-kidney/one-clip hypertension, male Lewis rats weighing 80—90 g were used and glomeruli were isolated 20 weeks after induction of hypertension. 4) The effect of dietary protein intake was followed in male Wistar rats (200 g) receiving diets containing casein fractions of 0.05, 0.20, or 0.60 for a period of 6 weeks. The diets were made isocaloric by addition of starch and animals were pairfed according to the 0.60 casein group. Isolation of glomeruli The animals were sacrificed under anaesthesia with hexobarbital (150 mg/kg) and the kidneys were obtained by a mid abdominal incision. The organs were dissected longitudinally, the medulla was excised and the remaining cortical tissue was minced. The resulting homogenate was passed through stainless steel sieves (250 μπι pore size) purchased from Linker, Kassel, Germany. The sieved material was suspended in ice-cold saline and poured on a 50 μπι nylon sieve (Schweizerische Seidengazefabrik, Zurich, Switzerland). This step allows cell debris and small fragments to pass through, while tubules and glomeruli are retained. Separation of glomeruli was achieved by passage through a 150 μπι nylon sieve which retains tubules and allows glomeruli to pass. The glomeruli were suspended in saline, gently spun at 400 £ and examined by light microscopy. The purity of glomeruli was 90—95%. Analytical methods Total proteolytic activity was measured with azocasein as substrate (Serva, Heidelberg, Germany) according to the method of Lange et al. (19). Briefly, 0.1 ml of the sample was incubated with 0.1 ml of a solution of azocasein (30 g/1) in Tris/HCl buffer for 45 min at 37 °C. The reaction was stopped by 0.5 ml of trichloroacetic acid (80 g/1). The samples were centrifuged at 500 g for 10 min and the released diazoamino acids were measured photometrically in the supernatant at 334 nm. The proteolytic activity was expressed in arbitrary U/mg protein with l arb. U representing a change of absorbance of 0.001/min. To study whether the breakdown of azocasein was pH-dependent, all proteolytic assays were performed at pH 5.4 and 7.4. In order to further characterize the azocaseinolytic activity, various proteinase inhibitors were used: 1) Phenylmethylsulphonylfluoride (Sigma, Munich, Germany) 2) fru7w-Epoxysuccinyl-L-leucylamido-butane (E-64; Sigma, Munich) 3) Ethylenediaminetetraacetate (EDTA; Sigma, Munich) Protein was determined according to the method of Lowry et al. (20), DNA according to the method of Maniatis et al. (21). The content of cathepsin B was measured by an enzyme-linked immunoassay according to Voller & Eidwell (22). In brief, flatbottomed microwell plates (Falcon, Heidelberg, Germany) were coated with rabbit anti-cathepsin B antibody (Serotec, Kildington, UK) by incubating wells with 0.1 ml of antibody diluted 1 : 1000 with carbonate/bicarbonate buffer for 18 hours at 4 °C. The antibody was poured off and unbound sites were blocked by incubation with 0.1 ml of bovine serum albumin (10 g/1). The sample (0.1 ml) was then added and incubated for 2 hours. Cathepsin B (Nova Biochem, Sandhausen, Germany) was used as standard. The supernatants were removed and the wells were washed thrice in NaCl/Tween. Subsequently, 0.1 ml of the anticathepsin B antibody conjugated with horseradish peroxidase (Serotec, Kildington, UK) diluted 1 :2000 was added to each well. After 2 hours the supernatants were removed and the substrate for horseradish peroxidase, 0-phenylenediamine dihydrochloride (Sigma, Munich, Germany), was added. The plates were again incubated for 10 min and the reaction was stopped by addition of 0.1 ml of 3 mol/1 H2SO4. The absorbance of each well was determined with an ELI S A reader (Behring, Marburg, Germany). Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 10

643

Schaefer et al.: Proteinases in glomerulosclerosis The activities of cathepsins B, H, and L were assayed as previously described by Barrett & Kirschke (23, 24) using fluorogenic peptidyl substrates (peptidyl-7-amido-4-methylcoumarins(-AMC); Bachern, Heidelberg, Germany): 7-Arg-Arg-AMC for cathepsin B, H-Arg-AMC for cathepsin H and Z-Phe-ArgAMC in combination with the specific cathepsin L inhibitor ZPhe-Phe-CHN2 for cathepsin L. In the case of cathepsin H, 0.1 mmol/1 puromycin dihydrochloride was used to inhibit arylamidases, which otherwise interfere by digesting H-Arg-AMC. For cathepsin L 0.6 μηιοΐ/l Z-Phe-Phe-CHN2 and 4 mmol/1 phenylmethylsulphonylfluoride were added to the reaction mixture. Cathepsin L catalytic activity was calculated as the difference in Z-Phe-Phe-AMC hydrolysis in the presence and absence of the cathepsin L inhibitor. The fluorescence of free 7-amino-4methylcoumarin was determined by excitation at 360 nm and emission at 460 nm with a Perkin-Elmer spectrofluorimeter. The activities of collagenase and gelatinase were measured fluorometrically according to Tschesche et al. (25) using rat tail collagen type-1 and denatured rat tail collagen typ-1, respectively. Collagenase measurements were carried out at 25 °C to prevent collagen denaturation. Gelatinase activity was determined in the presence of 4 mmol/1 phenylmethylsulphonylfluoride for inhibition of serine proteinases. After 18 h of incubation, the digested collagen fragments were labelled with fluorescamine (20 g/1). The fluorescence was measured by excitation at 390 nm and emission at 475 nm. Statistics Statistical analysis was performed by Student's t-test. Significance was defined as a p value of less than 0.05. All data are given as means ± SEM.

Results

Obese Zucker rats Proteolytic activity against azocasein in isolated glomeruli was maximal at pH 5.4, both in the obese and lean control animals. In comparison with lean animals, glomeruli from obese rats displayed lower proteolytic activities at both pH values (pH 5.4: 11.4 ± 0.7 vs 8.9 ± 0.4; pH 7.4: 9.3 + 0.6 vs 5.8 ± 0.7 arb. U/mg protein) (fig. 1). The same pattern emerged when the proteolytic activity was related to the DNA — rather than protein content (data not shown). In contrast, the glomerular content of cathepsin B was not related to the proteolytic activity at pH 5.4. The tissue mass of this enzyme was found to be higher in glomeruli from obese Zucker rats than in those from lean littermates (1.35 ± 0.13 vs 0.70 ± 0.11 μg/mg protein) (fig. 2).

r % ? ο α.

L

Control rats

Obese Zucker rats pH5.4

=£=

Control rats pH7.4

Obese Zucker rats

Fig. 1. Proteolytic activity in glomeruli isolated from obese Zucker rats and control animals. Mean values ± SEM from 8 animals in each group. * p < 0.05 for obese Zucker vs lean littermates. •E* 1.8 "05

I 15 ? "Si 1.2

I

0-8

§ ω 0.6

t 0,3

Control rats

Obese Zucker rats

Fig. 2. Cathepsin B content in glomeruli isolated from obese Zucker rats and control animals. Mean values ± SEM from 8 animals in each group. * p < 0.01 for obese Zucker vs lean littermates. Έ2° 'jo S 15

5* 5

Young rats

pH5.4

Old rats

Young rats pH7.4

Old rats

Fig. 3. Proteolytic activity in glomeruli from old and young control rats. Mean values ± SEM from 8 animals in each group. * p < 0.05 for old versus young animals.

The aging kidney Azocaseinolytic activity in glomeruli isolated from old (27 months) and young rats (6 months) was determined at pH 5.4 and pH 7.4. At both pH values the degradation of azocasein was reduced in the glomeruli from old animals (pH 5.4: 9.8 ± 0.8 vs 17.7 ± 0.8; pH 7.4: 6.4 ± 0.7 vs 11 ± 0.5 arb. U/mg protein) (fig. 3). The same held true when the proteolytic activity Eur. J. Clin. Chem. Clin. Biochem. / Vol. 30,1992 / No. 10

was related to the DNA content of the sample (data not shown). In order to further characterize these proteolytic activities, the effect of different proteinase inhibitors on the breakdown of azocasein was investigated. At pH 5.4 the cysteine proteinase inhibitor E-64 revealed a considerable inhibition of the glomerular proteinase activity both in young (62%) and

644

Schaefer et al.: Proteinases in glomerulosclerosis

old animals (48%), suggesting that the bulk of proteolytic activity is of the cysteine proteinase type (tab. 1). Tab. 1. Effect of different proteinase inhibitors on the glomerular proteinase activities Inhibitor

Concen- Residual activity tration fraction mmol/1 young rats old rats

None

1.00

1.00

Phenylmethylsulpho nyl fluoride (pH 7.4)

1

0.72

0.92

//YWs-Epoxysuccinyl-L-leucylamido-butane (pH 5.4)

1

0.38

0.52

Ethylenediamine-tetraacetate (pH 7.4)

5

0.86

0.93

LT> cn

o

-

l

Sf iam

OF)erate

*

*

o

Proteolytic activity [U/mg prot