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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 19, 2001 as doi:10.1096/fj.00-0493fje. |
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Departments of Internal Medicine and of Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany; Department of Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany, and Institutes of Molecular Biology and Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic
2Correspondence: Med. Poliklinik, Albert-Schweitzer-Str. 33, 48129 Muenster, Germany. E-mail: SchaefL{at}uni-muenster.de
SPECIFIC AIMS
The biological response towards transforming growth factor beta (TGF-ß), a key mediator of fibrotic diseases, is either directly or indirectly influenced by complex formation with members of the small leucine-rich proteoglycan (SLRP) family, of whom decorin has been used successfully as a therapeutic agent in experimental glomerulonephritis. By following the expression and immunolocalization of the four most well-known SLRPs (decorin, biglycan, lumican, fibromodulin), we sought to gain further insight into the mechanism of their potential anti-fibrotic role during different stages of human diabetic nephropathy.
PRINCIPAL FINDINGS
1. Increased transcription of decorin, biglycan, lumican,
and fibromodulin in renal tubulointerstitium and glomeruli during all
stages of diabetic nephropathy
Based on Northern blot analysis of total RNA from renal cortex and
on real-time RT-PCR from isolated glomeruli, an overexpression of all
four SLRPs was found in renal tissue from patients with diabetic
nephropathy as compared with age- and sex-matched healthy controls.
Up-regulation was most pronounced for decorin and biglycan, both in
whole renal cortex and in isolated glomeruli. During all stages of
diabetic nephropathy (incipient, manifest, and advanced) the four SLRPs
became induced, as shown by in situ hybridization, in the
tubulointerstitium and in glomeruli, regardless of the development of
nodular (Fig. 1B
)
or diffuse glomerulosclerosis (Fig. 1D
). Ectopic expression
of decorin and lumican was observed in epithelial cells of distal
tubules (not shown).
|
2. Discrepancy between glomerular expression and protein
accumulation of decorin, biglycan, lumican, and fibromodulin in human
diabetic nephropathy
As expected, an enhanced accumulation of all four SLRP proteins
was observed in the tubulointerstitium in all cases of diabetic
nephropathy. Most intense immunohistochemical stainings were found
around tubules in areas of interstitial fibrosis. In remarkable
contrast to the observations in the tubulointerstitium, the enhanced
glomerular expression of SLRPs was not mirrored by glomerular
accumulation of the respective proteoglycan core proteins. Biglycan,
decorin, lumican, and fibromodulin were almost undetectable in diabetic
glomeruli from kidneys with incipient or manifest nephropathy (Fig. 1A
). They were present only in glomeruli with advanced
sclerosis, where strong accumulation of all four SLRP proteins was
observed. This finding was most pronounced in scarred glomeruli with
obliteration of the urinary space and fibrous adhesion of the
glomerular tuft to Bowman’s capsule, in areas of fibrous organization
of the urinary space, and in the capsule of Bowman as well as
periglomerularly (Fig. 1C
), which correlated very well with
the distribution of type-I collagen.
3. Determination of decorin in urine and plasma from
patients with diabetic nephropathy
The discrepancy between glomerular mRNA and protein levels of
decorin, biglycan, lumican, and fibromodulin in diabetic nephropathy
could be explained by the assumption that these proteoglycans are not
retained at the site of their formation but are removed via the blood
stream and/or the urinary tract. Indeed, plasma concentrations of
decorin core protein were significantly (P < 0.001)
elevated in manifest diabetic nephropathy (2.09±0.17 ng/ml; GFR:
55.3±7.7 ml/min; mean ± SE; n=6)
when compared with nondiabetic control patients (0.89±0.06 ng/ml,
n=10). A further increase occurred in advanced nephropathy:
(3.18±0.36 ng/ml; GFR: 11.3±0.9 ml/min; mean ±SE;
n=6). No significant difference in plasma concentrations of
decorin was found between control subjects and patients with incipient
diabetic nephropathy. Urinary decorin excretion became detectable only
in advanced diabetic nephropathy and reached values between 1.6 and 40
µg/l. It is of note that urinary excretion of decorin did not simply
correlate with proteinuria because, in urine samples from patients with
nephrotic-range proteinuria and normal renal function, decorin was not
detectable.
4. Determination of urinary decorin/TGF-ß1 complexes
Urinary TGF-ß1/decorin complexes were quantitated indirectly by
measuring the difference in soluble TGF-ß1 concentrations after
treating the urine with control IgG and anti-decorin IgG, respectively.
Whereas in nondiabetic control patients 1.2±0.3 ng of TGF-ß1/24 h
were excreted as complex with decorin, excretion rose to 2.0±0.5 ng/24
h in incipient (P > 0.05), 4.2±1.1 ng/24 h (P
< 0.05) in manifest, and to 9.1±2.3 ng/24 h (P < 0.05) in
advanced diabetic nephropathy, respectively. In all groups of patients,
decorin-bound TGF-ß1 represented about 6% of the total amount of the
excreted cytokine.
CONCLUSIONS
This report is the first to show that the expression of decorin,
biglycan, lumican, and fibromodulin is enhanced greatly in glomeruli
from human diabetic kidneys at different stages of nephropathy but that
this overexpression is not mirrored by glomerular accumulation of the
respective proteins, except in advanced nephropathy. Overexpression of
decorin has been reported before in the cortex of
streptozotocin-diabetic mouse kidneys as well as in cultured mesangial
cells under high-glucose conditions, resulting in increased
proteoglycan production. Additionally, the presence of collagen type-I
within the mesangial matrix has shown to be necessary for binding
intravenously administered decorin as well as for binding decorin to
renal tissue sections in vitro. This finding led us to
formulate the hypothesis that in diabetic kidneys the bulk of newly
synthesized small proteoglycans is not retained within the glomerular
matrix but is removed via the vasculature and/or the urinary tract
(Fig. 2
).
|
In the plasma of healthy individuals decorin is present in low
concentrations. In diabetic nephropathy, decorin plasma levels rose in
those patients who suffered from a progressive decline in their
glomerular filtration rates. At earlier stages of the disease,
increased plasma levels of decorin could not be recognized with the
present methodology. However, decorin and possibly also the other small
proteoglycans are efficiently cleared from the circulation.
Intravenously injected decorin has been shown to become removed by the
liver, possibly by the hyaluronan- and galactosaminoglycan-recognizing
scavenger receptors of liver endothelial cells (Fig. 2)
. Cultured
macrovascular endothelial cells have also been shown to take up decorin
by receptor-mediated endocytosis. In addition, we have recently been
able to detect decorin in the glomerular basement membrane and in
vacuolar structures of normal human proximal tubular epithelial cells
(Fig. 2)
, which do not express decorin. These findings
support the hypothesis that an increased secretion of small
proteoglycans by diabetic glomeruli is compensated in part by an
increased rate of endocytosis and that significant decorin levels in
the urine are observed only when the filtered load overcomes the
tubular capacity for reabsorption.
It had been demonstrated that decorin treatment, either by proteoglycan
application or by gene transfer, exerts beneficial effects in fibrotic
disorders with TGF-ß overproduction in the kidney and in other organs
as well. The pathophysiological mechanism behind this effect, however,
remained unclear because complex formation with decorin was found
either to inhibit or activate or not to influence the cytokine’s
activity at all. Using a model of cell-populated collagen lattices, we
could show that decorin could sequester TGF-ß in the extracellular
matrix, which subsequently resulted in a reduced biological activity of
the cytokine. Similar arguments may explain the findings of the present
study. Enhanced proteoglycan production may result in removal of
TGF-ß from the mesangial matrix as long as there is only a small
amount of proteoglycan-binding proteins present in the tissue (Fig. 2)
.
Only in advanced diabetic nephropathy, especially in scarred glomeruli,
decorin deposition was found in areas of fibrous formations where it
co-localized with deposits of type-I collagen. This process may reflect
a new regulatory mechanism by which decorin modulates the evolution of
TGF-ß-mediated renal fibrosis in a way that a ternary complex of
decorin, type-I collagen, and TGF-ß withdraw the cytokine from its
cell surface receptors, thereby indirectly signaling that sufficient
matrix has been synthesized and that TGF-ß gene expression should be
turned off (Fig. 2)
.
In summary, from the results of this investigation it is tempting to speculate that in the diabetic kidney increased quantities of glomerular proteoglycans are synthesized but not retained in the mesangial matrix. Instead, these proteoglycans are removed via glomerular capillaries or the urinary tract, in part as complexes with TGF-ß. This specific process may counteract the vicious circle of increased TGF-ß production and enhanced matrix deposition in diabetic nephropathy.
FOOTNOTES
1 To read the full text of this article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0493fje ; to cite this
article, use FASEB J. (January 19, 2001)
10.1096/fj.00-0493fje 
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