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Pathogenesis of Diabetic
Nephropathy: Biochemical
and Functional Alterations of
Glomerular Basement
Membrane and Mesangium
BENITO A. YARD, NICOLE F. VAN DET AND
FOKKO J. VAN DER WOUDE
INTRODUCTION
Changes in the composition of the mesangium and the glomerular basement
membrane (GBM) in diabetic nephropathy are thought to be responsible, at
least in part, for the functional changes observed. Although these functional
changes are discussed more extensively in Chapters 2 and 6, the current
clinical classification of nephropathy (1) will be briefly recapitulated first in
order to be able to put the biochemical and morphological alterations in
context.
The classification of nephropathy into several distinct phases can be used
in IDDM and NIDDM. Initial changes include glomerular hyperfiltration and
hyperperfusion. A silent phase follows hyperfiltration. It has been documented that, at least in IDDM patients, there are already subtle morphological
changes present during this phase, such as thickening of the glomerular
basement membrane, glomerular hypertrophy, mesangial expansion and
Diabetic Nephropathy. Edited by C. Hasslacher.
# 2001 John Wiley & Sons, Ltd.
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with different composition, the lamina rara interna and externa. The
lamina rara externa contains heparan sulphate proteoglycan (HSPG) and
attachment proteins such as laminin, entactin and other glycoproteins. The
structure and composition of the GBM is of importance for its function as a
filtration barrier in the glomerulus. Mesangial cells (MC) are embedded in
an ECM between the capillaries and play a critical role in the modulation of
glomerular blood flow and filtration by contraction and relaxation. The
mesangial matrix, although developmentally and morphologically distinct
from the GBM, is composed of essentially the same components, i.e. collagen type IV (a1, a2), laminin and HSPG. Furthermore, MC have been
found to produce fibronectin, entactin, collagen V and VI. Since in diabetic
nephropathy most data have been reported on abnormalities in glomerular
collagen type IV, laminin, fibronectin and HSPG content, these compounds
will now be discussed.
COLLAGEN TYPE IV
Collagen was one of the first known constituents of the extracellular matrix;
collagen type IV provides a scaffold for other ECM components, due to its
network-like structure. Klein et al (11) reported that diabetic glomeruli had
greater hydroxyproline content than non-diabetic glomeruli when content
was expressed per glomerulus (21.9 3.3 ng vs. 7.1 0.5 ng) and when
expressed per mg dry weight of glomeruli (44.0 2.4 mg vs. 31.6 1.9 mg).
Glomeruli from diabetics of longest duration show the greatest increases in
mass and hydroxyproline values. A pathologist's semiquantitative estimation of diffuse glomerulosclerosis revealed a high correlation between
hydroxyproline values and histologic determination of the extent of the
renal lesion. After the identification of the different types of collagen type
IV chains, it became clear that there is a distinct distribution of the various
chains in normal and diabetic GBM and mesangial matrix. The first study
addressing this issue was reported by Kim et al (12). They showed that
during the course of the disease, the distribution of a3(IV) segregated from
that of a1(IV). In diabetic kidneys, antibodies to a3(IV) reacted intensely
with the thickened GBM but not with the mesangium. In contrast, the
reactivity of antibodies to various components of a1(IV) was prominent
within the expanded mesangial matrix, with significant decrease in reactivity in the peripheral capillary wall. These findings have been confirmed in
IDDM (13) as well as NIDDM (14) and suggest that there might be different
sites of synthesis [a1 (IV) collagen from endothelial/mesangial cells and
alpha 3(IV) chains from GVEC], independent control mechanisms and/or
differences in degradation. An increase of type IV collagen has also been
described in glomeruli of streptozotocin-treated C57BL/SJL mice, which
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develop histological lesions closely resembling human diabetic glomerulosclerosis (15). There were no glomerular lesions in diabetic mice transgenic
for a growth hormone analogue that competes with native GH and results in
dwarfism. Enhanced synthesis of collagen in non-renal tissues from IDDM
patients with nephropathy has also been suggested (16,17). Trevisan et al
(16) studied overall collagen metabolism and total protein synthesis in
either normal (5 mM) or high (25 mM) glucose concentrations from 14 insulin-dependent diabetic (IDDM) patients with nephropathy, 14 IDDM
patients without nephropathy and 14 healthy subjects. In high glucose
concentrations (25 mM), overall collagen synthesis (measured as [3H]proline incorporation into extracellular and intracellular collagenase-sensitive
material) was significantly greater in the patients with nephropathy than in
the patients without nephropathy or in healthy control subjects. These
findings were confirmed in an in vivo study (17) in which skin basement
membrane from IDDM patients with and without nephropathy was studied
immunohistologically. Increased staining was found for collagen type I and
advanced glycosylation end products in patients with diabetic nephropathy.
The skeletal muscle capillary basement membranes of IDDM patients with
diabetic nephropathy were also shown to have an increased collagen type
IV content (18).
Since the increased collagen content of various tissues in diabetic nephropathy in diabetic nephropathy is so well established, many investigators
have sought to find a clue to pathogenesis by studying collagen synthesis
and turnover. A Danish group looked at the genetic variation of a collagen
IV a1-chain polymorphism in IDDM patients and found no association to
nephropathy (19). Attempts to study the interaction between high glucose
concentrations and collagen synthesis have been more successful: mesangial cells growing in high glucose concentrations produce more collagen
types I and IV, and also more transforming growth factor-beta (TGF-b). It is
now believed that TGF-b, a known prosclerotic cytokine, mediates the
stimulation of ECM production in mesangial cells by high glucose in an
autocrine fashion. Addition of neutralizing anti-TGF-b antibody, but not
normal rabbit IgG, significantly reduced the high glucose-stimulated incorporation of 3[H]proline in mesangial cells (20). Denaturing SDSPAGE
revealed that mainly collagen types I and IV were stimulated by high
(450 mg/dl) d -glucose. This high glucose-mediated increase in collagen
synthesis, as well as increased levels of mRNA encoding a2(I) and a1(IV),
could be reduced with anti-TGF-b antibody. Decreased mesangial cell
growth and increased collagen gene expression can be induced not only
by high glucose concentrations but also by increased concentrations of
glycated proteins. Amadori glucose adducts in glycated albumin
inhibited mesangial cell [3H]-thymidine incorporation; this effect could be
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44
CO2
O
H
OH
H
NH
45
R = SO3 /H
OH
OR
C
CH3
O
N-acetyl glucosamine glucuronic acid
CH2OR
H
CO2
O
H
OH
H
NH
OH
OR
SO3
N-sulphate glucosamine
CH2OR
H
O
H
H
OH
H
glucuronic acid
H
O
NH
CO2
O
O
OH
OR
SO3
N-sulphate glucosamine
Figure 3.1
1.
2.
3.
iduronic acid
A decreased 35 S-sulphate incorporation into glomerular basement membranes was found by several groups (38,39). Kanwar et al (40) reported
decreased de novo synthesis of glomerular proteoglycans in diabetic rats,
and Rohrbach et al (41,42) found changes in, and reduced synthesis of,
basement membrane HSPG in diabetic mice. Parathasarathy and Spiro
(43) obtained kidneys of patients with diabetes at autopsies. Histological
hallmarks of glomerulopathy were present in these specimens. They
observed, using biochemical techniques, that the GBM of patients with
DN contained fewer GAGs than kidneys of non-diabetic controls. A few
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years later, their early report was confirmed (44). Deckert and co-workers
put forward an hypothesis in which altered HSPG metabolism due to
hyperglycaemia plays a pivotal role in the pathogenesis of diabetic nephropathy (45). A genetic heterogeneity for a key-enzyme in HS modification, i.e.
N-deacetylase/N-sulphotransferase (DAST), would result in an increased
vulnerability to high glucose levels in a subset of patients. This would lead
to an undersulphation of HS. Resultant loss or reduced synthesis of HSPG in
the GBM could provide a molecular basis for albuminuria, an early sign of
diabetic nephropathy (4547). To date, two cDNAs encoded by an 8 kb and
4 kb transcript, designated as DAST-1 and -2, respectively, have been
identified (48,49). These two enzymes exhibit alternate specificities that
lead to varying extents of N-sulphation. Whereas DAST-1 generates
approximately 40% N-sulphation of HS, DAST-2 produces approximately
80% N-sulphation of HS (50). Glomerular N-deacetylase activity is inhibited
in diabetic rats, as has been described by Koefoed-Enevoldsen (51), and thus
may play an important pathogenic role in DN. It does not, however, explain
why only approximately 30% of diabetic patients develop DN. Thus far, no
polymorphism in the DAST gene loci has been reported. Decreased mesangial HSPG, with the resulting lack of inhibition of extracellular matrix
production and of cellular proliferation, could contribute to mesangial
expansion, a hallmark of nephropathy with prognostic value (52). Furthermore, regarding albuminuria as a marker for generalized vascular damage,
changes of HS could be involved in altered lipoprotein lipase (LPL)mediated lipid metabolism. Therefore, changes in HSPG metabolism
induced by hyperglycaemia could be of pathogenic relevance in the development of both glomerulosclerosis and arteriosclerosis, a frequently
observed combination in patients with DN. In fact, generalized loss of
HSPG may also lead to an increase in vascular smooth muscle cell proliferation in the vessel and may result in arteriosclerosis. Recent studies have
further explored HSPG expression in human diabetic glomerulopathy. HS
and HSPG were found to be decreased (13,53,54) in patients with overt
diabetic nephropathy, using newly developed monoclonal antibodies with
reactivity to HS and HSPG core proteins (53). A decreased expression of
GBMHS without changes in HSPG core protein staining was found, suggesting a vulnerability in the metabolism of the negatively charged side
chain of HSPG in DN. Now that it has become evident that agrin, but not
perlecan, is the major HSPG present in the GBM (55), these studies have to
be re-evaluated to exclude an altered expression of agrin in DN. The expression of agrin in DN has not been studied so far. Vernier et al (56) described a
correlation between GBM HSPG expression and mesangial expansion, using
electron microscopy morphometry and histological staining techniques.
Thus, recent studies confirmed and visualized the results from the Parathasarathy and Spiro Study (43). Furthermore, they suggest that especially a
47
48
49
50
Podocytes
Mesangial cells
51
Increased
TGF- production
Overall increased cell
proliferation
Altered extracellular
matrix synthesis
Proteinuria and mesangial
expansion
Figure 3.2 Synopsis of the possible pivotal role of HSPG in the pathogenesis of the
glomerular alteration in diabetic nephropathy. There are insufficient data on
glomerular endothelial cells in this respect and these cells are therefore not depicted
in this diagram
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patients with diabetic nephropathy plays a pivotal role in both diabetic nephropathy and macroangiopathy (mesangial cells and vascular smooth muscle cells can be regarded as phenotypically related),
leading to mesangial and smooth muscle cell perturbations, an altered
permeability of glomerular and vessel walls and a disturbance of the
physiological modulatory influence of HSPGs on growth factors such
as ANG II, TGF-b and bFGF.
The logical consequence of this line of thought is to treat experimental animals and humans with diabetic nephropathy with heparinoids. Both experimental animal studies (92) and preliminary
clinical trials (93,94) have been published, showing favourable effects
of HSPG or heparin preparations. Further studies with newer, betterdefined compounds are necessary to enable us to attain a better
understanding of the mode of action to document and reduce possible
side-effects.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
53
54
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
55
Tisher CC, Hostetter TH. Diabetic Nepropathy. In Renal Pathology, Tisher CC,
Brenner BM (eds). Lippincott: Philadelphia, PA, 1991; 1387412.
Hostetter TH. Diabetic Nephropathy. In The Kidney, Brenner BM, Rector FC
(eds). WB Saunders: Philadelphia, PA, 1991; 1695727.
Dixon J, Loftus SK, Galdwin AJ et al. Cloning of the human heparan sulfate-Ndeacetylase/N-sulfotransferase gene from the Treacher Collins syndrome candidate region at 5q32q33.1. Genomics 1995; 26: 2394.
Kusche-Gullberg M, Eriksson I, Pikas DS, Kjellen L. Identification and expression of two heparan sulfate glucosamyl N-deacetylase/n-sulfotransferase
genes. J Biol Chem 1998; 273: 119027.
Rosenberg RD. Heparan sulfate proteoglycans of the cardiovascular system. J
Clin Invest 1997; 99: 206270.
Kofoed-Enevoldsen A. Inhibition of glomerular glucosamyl N-deactetylase in
diabetic rats. Kidney Int 1992; 41: 10218.
Mauer SM. Structuralfunctional correlations of diabetic nephropathy. Kidney
Int 1994; 45: 61222.
Van den Born J, van den Heuvel LPWJ, Bakker MAH et al. Monoclonal antibodies
against the protein core and glycosaminoglycan side chain of glomerular basement membrane heparan sulfate proteoglycan: characterization and immunohistological application in human tissues. J Histochem Cytochem 1994; 42: 89102.
Van den Born J, van den Heuvel LPWJ, Bakker MAH et al. Distribution of GBM
heparan sulfate proteoglycan core protein and side cahins in human glomerular
diseases. Kidney Int 1993; 43: 45463.
Groffen AJ, Ruegg MA, Dijkman H et al. Agrin is a major heparn sulfate
proteoglycan in the human glomerular basement membrane. J Histochan Cytochem 1998; 46: 19.
Vernier RL, Steffes MW, Sisson-Ross S, Mauer M. Heparan sulfate proteoglycan
in the glomerular basement membrane in type I diabetes mellitus. Kidney Int
1992; 41: 107080.
Wasty F, Alavi MZ, Moore S. Distribution of glycosaminoglycans in the intima
of human aortas: changes in artherosclerosis and diabetes mellitus. Diabetologia
1993; 36: 31622.
Yaoita E, Oguri K, Okayama E. Isolation and characterization of proteoglycans
synthesized by cultured mesangial cells. J Biol Chem 1990; 265: 52231.
Groggel GC, Hovingh P, Linker A. Proteoglycan and glycosaminoglycansynthesis by cultured rat mesangial cells. J Cell Physiol 1991; 147: 4559.
Thomas GJ, Mason RM, Davies M. Characterization of proteoglycans
synthesized by human adult mesangial cells in cultures. Biochem J 1991; 277:
818.
Klein DJ, Oegema TR, Fredeen TS et al. Partial characterization of proteoglycans
synthesized by human glomerular epithelial cells in culture. Biochem Biophys
1990; 277: 389401.
Stow JL, Soroka CJ, Mackay K, Striker L, Farquhar MG. Basement membrane
heparan sulfate proteoglycan is the main proteoglycan synthesized by glomerular epithelial cells in culture. Am J Pathol 1989; 135: 63746.
Olgemoller B, Schwaabe S, Gerbitz KD, Schleicher ED. Elevated glucoses
decrease the content of a basement membrane associated heparan sulfate
proteoglycan in proliferating porcine mesangial cells. Diabetologia 1992; 35:
1836.
Silbiger S, Schlondorff D, Crowley S et al. The effect of glucose on proteoglycans
produced by cultured mesangial cells. Diabetes 1993; 42: 1852.
56
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
86.
87.
88.
89.
90.
91.
92.
93.
94.
57