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Original Studies |
University of Melbourne, Department of Medicine, and Endocrinology Unit (A.A., S.P., G.J.), Austin and Repatriation Medical Center, Heidelberg, Victoria 3084, Australia
Address all correspondence and requests for reprints to: Dr. Leon A. Bach, University of Melbourne, Department of Medicine (Austin Campus), Austin and Repatriation Medical Center, Studley Road, Heidelberg, Victoria 3084, Australia. E-mail: bach{at}austin.unimelb.edu.au
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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IGF-I and IGF-II are widely expressed peptides that are essential for normal development (2). The actions of IGF are modulated by a family of at least six specific, high affinity binding proteins (IGFBPs) (2, 3). At a cellular level, IGFBPs may inhibit or potentiate IGF actions (2, 3). In serum, IGFs are predominantly found within 150-kDa ternary complexes that contain IGFBP-3, an approximately 40-kDa N-glycosylated protein that binds IGFs with high affinity, and an 85-kDa acid-labile subunit (4). IGFs within the 150-kDa complex remain in the vascular compartment, serving as a potential reservoir of IGFs while protecting the host from possible acute insulin-like effects of free IGFs. Circulating IGFs also bind to IGFBPs in binary complexes that are able to leave the vascular compartment. Binary, but not ternary, complexes are found in tissues.
Limited proteolysis of IGFBPs has recently emerged as a physiological mechanism by which the IGF binding affinities of IGFBPs are reduced (5). This may lead, in turn, to increased availability of IGFs for binding to IGF-I receptors. A wide range of IGFBP proteases, including serine and aspartic and metalloproteases, have been described. Increased IGFBP-3 proteolysis has been reported in serum from pregnant women and patients with catabolic conditions, including diabetes (6, 7).
IGF-I and IGFBP-1 to -6 are all expressed in normal rat kidney in distinct distributions (8, 9). A number of roles for the IGF system in renal development and normal physiology have been described (10). Humans and animals deficient in GH (and therefore IGF-I) have decreased kidney size, glomerular filtration rate, and renal plasma flow, and infusion of IGF-I increases these parameters.
The IGF system has been implicated in animal models of diabetic nephropathy (11). Kidney enlargement during the early stage of experimental diabetes is associated with transiently increased IGF-I levels (12, 13), whereas renal IGF-I receptor levels are increased for a more sustained period (14). Changes in levels of renal IGFBPs have also been described in diabetic rats (15, 16, 17, 18). However, there have been few studies of the role of the IGF system in human diabetic nephropathy. Serum IGF-I levels are not substantially altered in diabetic nephropathy (11), but these may not reflect local renal changes, which may be significant because the IGF system has important paracrine/autocrine actions. Indeed, this is exemplified by diabetic adolescents with microalbuminuria who had higher urinary, but not plasma, IGF-I levels than those with normoalbuminuria (19).
As urinary IGFBPs are altered in some, but not all, kidney diseases (20, 21), we hypothesized that the development of diabetic nephropathy might be associated with changes in urinary IGFBPs. We found that intact IGFBP-3 levels were decreased due to increased proteolysis in the urine of patients with diabetic nephropathy.
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Subjects and Methods |
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Urine was examined from diabetic patients attending the Endocrinology Clinic at the Austin and Repatriation Medical Center with noninsulin dependent diabetes mellitus (NIDDM; n = 34; age, 55.9 ± 8.8 yr, mean ± SD), without a urinary tract infection or history of other kidney diseases. The diagnosis of NIDDM was defined by clinical characteristics: age of diagnosis more than 35 yr, no history of ketoacidosis, and no absolute insulin requirement. Nephropathy was classified by albumin excretion rate (AER; normoalbuminuria, <20 µg/min; microalbuminuria, 20–200 µg/min; macroalbuminuria, >200 µg/min). To be classified as micro- or macroalbuminuric, AERs had to be elevated on at least two of three occasions.
We also examined urine from a smaller number of patients with insulin-dependent diabetes mellitus (IDDM; n = 14; age, 40.8 ± 11.8 yr) without a urinary tract infection or history of other kidney diseases. The diagnosis of IDDM was defined by a history of ketoacidosis and/or absolute insulin requirement.
Nine healthy control subjects (n = 9; age, 37.6 ± 14.5 yr) without a urinary tract infection, hypertension, or history of other kidney diseases were also studied.
Reagents
IGF-II was a gift from Eli Lilly & Co. (Indianapolis, IN). Nonglycosylated recombinant human IGFBP-3 was a gift from Dr. C. Maack, Celtrix Pharmaceuticals, Inc. (Santa Clara, CA). Rabbit antihuman IGFBP-3 antiserum was a gift from Dr. Janet Martin, Sydney University (Sydney, Australia). Biotinylated goat antirabbit antibody and streptavidin-horseradish peroxidase conjugate were purchased from Amersham Pharmacia Biotech (Castle Hill, Australia). Protease inhibitors [ethylenediamine tetraacetate (EDTA), 1,10-phenanthroline, leupeptin, aprotinin, and pepstatin] were purchased from Sigma (St. Louis, MO).
Preparation of urine samples
First voided morning urine samples or aliquots from 24-h urine collections were centrifuged at 1200 rpm for 10 min at 4 C to separate cellular and other sediments. Supernatants were stored at -20 C for up to 2 weeks before dialysis. Stored urine specimens were thawed slowly and centrifuged at 2500 rpm for 10 min at 4 C, and the pellet was discarded. Supernatants were dialyzed three times against distilled water for 24 h at 4 C using 12,000–14,000 molecular mass cut-off membranes (Union Carbide, Chicago, IL). Aliquots (1 mL) were freeze-dried and stored at -20 C.
Iodination of IGF-II and recombinant human IGFBP-3
IGF-II and IGFBP-3 were iodinated by the chloramine-T method to specific activities of 150–200 and 15 µCi/µg, respectively, as previously described (22).
Western ligand blotting (WLB)
Dialyzed urine samples (1 mL original volume) were diluted with nonreducing sample buffer (0.5 mol/L Tris, pH 6.8; 10% glycerol; 2% SDS; and 0.05% bromophenol blue) and loaded onto a 0.75-mm discontinuous SDS-12% PAGE at 200 V for 1.5–3 h. Electrophoresed proteins were electroblotted onto a nitrocellulose membrane that was blocked with 1% BSA, incubated in buffer containing [125I]IGF-II (1.0 x 106 cpm), washed, and exposed to BioMax x-ray film (Eastman Kodak Co., Rochester, NY) for 5–7 days.
Western immunoblotting (WIB)
WIB was performed as previously described (22), using enhanced chemiluminescence with minor modifications. Dialyzed urine samples were separated by SDS-12% PAGE under nonreducing conditions and then transferred to nitrocellulose membranes as described above. Alternatively, membranes used for WLB were incubated in 10 mmol/L Tris-HCl and 150 mmol/L NaCl (pH 7.4) containing 3% Nonidet P-40 for 1 h at room temperature to remove bound ligand. Membranes were blocked in Tris-buffered saline with 5% nonfat dry milk and incubated with a polyclonal rabbit antihuman IGFBP-3 antibody (1:3000). Membranes were then successively incubated with biotinylated antirabbit Ig and streptavidin-horseradish peroxidase conjugate followed by detection using enhanced chemiluminescence (Super Signal, Pierce Chemical Co., Rockford, IL).
IGFBP-3 proteolysis assay
To further examine the presence of proteolytic activity toward
IGFBP-3, urine samples collected from healthy controls and
diabetic patients were analyzed according to the method of Lamson
et al. (23). Digestion mixtures were made up to a total
volume of 20 µL. 125I-Labeled nonglycosylated
IGFBP-3 (
30,000 cpm) was suspended in phosphate-buffered saline with
0.5 mmol/L calcium chloride and incubated for 5 h at 37 C with 2.5
µL serum or 50-fold concentrated urine (250 µL original volume) in
the absence of protease inhibitors or in the presence of EDTA (5
mmol/L), 1,10-phenanthroline (5 mmol/L), aprotinin (50 U/mL), pepstatin
(2.5 µg/mL), or leupeptin (5 mmol/L). Laemmli sample buffer was added
to stop digestion before separation by SDS-12% PAGE. Gels were fixed
with 10% acetic acid for 15 min, dried, and subjected to
autoradiography for 24 h. The amounts of low molecular mass bands
and intact tracer in each lane were determined by laser densitometry
(model 2222–020, Ultro Scan laser densitometer, LKB,
Bromma, Sweden). Results were corrected for tracer degradation by
subtracting the amount of lower molecular mass bands in control samples
containing tracer alone, and were expressed as a percentage of the
total optical density per lane that occurred in lower molecular mass
bands representing proteolytic cleavage products rather than intact
IGFBP-3.
Urinary albumin excretion
The urinary albumin concentration was measured in aliquots of 24-h urine specimens using immunoturbidimetry (Turbitimer, Behringwerke AG, Marburg, Germany). Intra- and interassay coefficients of variation were 4.5% and 3.0%, respectively (24).
Statistical analysis
Results are shown as the mean ± SD. Differences among diabetic patients with normo-, micro-, and macroalbuminuria were compared using one-way ANOVA, followed by Fisher’s protected least significant difference test for post-hoc comparisons. Relationships between the extent of IGFBP-3 proteolysis and other variables were examined both by simple regression and by stepwise multiple linear regression. AER data were log-transformed before regression analysis.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Patients with NIDDM were grouped according to degree of
albuminuria: normoalbuminuria (n = 9; AER, <20 µg/min),
microalbuminuria (n = 13; 20–200 µg/min), and macroalbuminuria
(n = 12; AER, >200 µg/min; Table 1
). Groups did not significantly differ
in age, duration of diabetes, renal function, blood pressure, or
treatment modality. There was a marginal difference between groups in
body mass index (P = 0.054).
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. Patients with macroalbuminuria were
older than those in other groups (P = 0.02). Patients
with nephropathy had a greater prevalence of hypertension
(P = 0.02). There were nonsignificant trends toward
increased duration of diabetes (P = 0.07), lower
creatinine clearance (P = 0.06), and higher systolic
blood pressure (P = 0.052) in patients with
nephropathy, although these findings must be interpreted with caution
given the small number of patients.
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Figure 1A shows a typical pattern of
IGFBPs in urine (lanes 1, 2, 4, and 6) and in serum (lanes 3, 5, 7, and
8) of control and diabetic patients as demonstrated by WLB. In control
serum (lane 8), the predominant band was 40–46 kDa, with less intense
bands of 24–35 kDa. In control urine (lane 1) the predominant band was
approximately 35 kDa, with other bands of 40–46 and 26 kDa. The
patterns of IGFBPs in serum from diabetic patients with
normoalbuminuria (lane 3), microalbuminuria (lane 5), and
macroalbuminuria (lane 7) were similar to that seen in control serum.
The pattern of urinary IGFBPs in normoalbuminuric diabetic subjects was
also similar to that in control subjects. In contrast, urine from
patients with micro- (lane 4) and macroalbuminuria (lane 6) lacked the
40- to 46-kDa band, and an approximately 18-kDa band was the most
predominant IGFBP in the latter sample.
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). This confirmed that the 40- to 46-, 30-, and
18-kDa bands were forms of IGFBP-3. Urine from micro- and
macroalbuminuric patients (lanes 4 and 6) contained less intact 40- to
46-kDa IGFBP-3, whereas levels of 18-kDa IGFBP-3, presumably a
fragment, were increased. The 18-kDa IGFBP-3 band was not seen in serum
from the same subjects. The 35-kDa band seen by WLB was identified as
IGFBP-2 by immunoblotting (results not shown).
There was substantial variability in the forms of IGFBP-3 present in
the urine of control subjects (Fig. 2A).
Nevertheless, 40- to 46-kDa intact IGFBP-3 was present in significant
amounts in 5 of 6 samples and was still detectable in the other (lane
4). Substantial amounts of intact IGFBP-3 were also present in 15 of 17
normoalbuminuric diabetic urine samples (e.g. Fig. 2B, lanes
1 and 2), but IGFBP-3 was reduced or absent in all 26 micro-
(e.g. lanes 3 and 4) or macroalbuminuric (e.g.
lane 5) urine samples. These observations were true regardless of
whether patients had NIDDM or IDDM.
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To investigate whether proteolysis of IGFBP-3 was altered in the
urine of patients with diabetic nephropathy, urine samples were
incubated with 30-kDa nonglycosylated
[125I]IGFBP-3 and analyzed by SDS-PAGE (Fig. 3). After incubation for 24 h at 37
C with micro- or macroalbuminuric diabetic urine samples, little or no
intact tracer remained, and the intensity of lower molecular mass bands
was increased (lanes 4 and 5). In contrast, the tracer was only
partially proteolyzed in the presence of urine from a normoalbuminuric
diabetic (lane 3) or a control (lane 7) subject. Only minimal
proteolysis was seen in the presence of serum from normal or diabetic
subjects (lane 2 and results not shown). These findings suggest that
proteolysis of IGFBP-3 is largely confined to the renal system in
patients with diabetic nephropathy. The proteolysis observed is not a
postvoiding phenomenon, because there was no difference in the urinary
IGFBP-3 patterns between samples stored immediately at -20 C and those
left at room temperature for 24 h as assessed by immunoblotting
(results not shown).
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). Levels of proteolysis of
IGFBP-3 in the healthy control group and the normoalbuminuric group
were 49 ± 15% and 29 ± 9%, respectively. The activities
in the microalbuminuric and macroalbuminuric groups were significantly
increased to 75 ± 25% and 84 ± 14%, respectively
(P = 0.0001).
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The extent of IGFBP-3 proteolysis was correlated with AER in patients
with NIDDM (r = 0.61; P = 0.0001; Fig. 4B and
Table 3). A similar relationship was
observed in patients with IDDM (r = 0.56; P =
0.04; Table 3). When IDDM and NIDDM patients were combined, there was
also a positive correlation between the extent of IGFBP-3 proteolysis
and the duration of diabetes (r = 0.33; P = 0.02;
Table 3) and a negative relationship with creatinine clearance
(r = 0.31; P = 0.03; Table 3). After stepwise
regression analysis, a final equation was derived, including
significant positive associations with AER and duration of diabetes and
an inverse association with age in NIDDM patients alone (overall:
r = 0.75; P < 0.0001) and in NIDDM and IDDM
patients combined (overall: r = 0.69; P <
0.0001). Stepwise regression analysis was not performed with IDDM
patients alone because of the small sample size. There was no
correlation between IGFBP-3 proteolysis and blood pressure or
hemoglobin A1c by either simple or stepwise
regression analysis.
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Figure 5 shows degradation of
recombinant human [125I]IGFBP-3 by urine from a
macroalbuminuric subject incubated for 5 h at 37 C with (lanes
3–7) and without (lane 2) protease inhibitors. Aprotinin (lane 6; a
serine protease inhibitor) and leupeptin (lane 7; a serine and thiol
protease inhibitor) inhibited degradation of IGFBP-3. In contrast,
pepstatin (lane 3; an aspartic protease inhibitor), EDTA (lane 4; a
chelator of divalent cations), and 1,10-phenanthroline (lane 5; a
matrix metalloproteinase inhibitor) did not inhibit IGFBP-3
degradation. Leupeptin and aprotinin inhibited IGFBP-3 proteolysis
to similar extents in urine samples from five subjects. After
incubation with leupeptin and aprotinin, proteolysis levels were
36 ± 24% and 36 ± 20%, respectively, compared with
70 ± 19% in untreated samples.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Previous studies have shown the presence of intact IGFBP-2 (35
kDa) and IGFBP-3 (40 kDa) as well as smaller IGFBPs in urine from
normal subjects (20). The IGFBP-2/IGFBP-3 ratio was higher in urine
than in serum, a finding confirmed in the present study. More detailed
studies showed proteolysis of IGFBP-3 in urine from control subjects
(21, 25). Using the same assay as that used in the current study, the
extent of IGFBP-3 proteolysis was 10–50% in these studies. One study
showed that the predominant form of IGFBP-3 in urine from children and
adults was 17.7 kDa in size (26); this form may have been missed or
understated in studies using WLB for detection, because this fragment
binds IGFs poorly.
Urinary IGFBPs have also been studied in a variety of kidney diseases. In patients with glomerulonephritis but normal renal function, urinary intact IGFBP-3 levels were increased (20). The urine of children with nephrotic syndrome contains increased amounts of intact as well as fragmented IGFBP-3, as determined by size exclusion chromatography (27). Another study showed increased IGFBP-3 proteolysis in urine from patients with Alport’s syndrome, but not in those with IgA nephropathy, systemic lupus erythematosus, or focal segmental glomerulosclerosis (21). In patients with acute or chronic renal failure (serum creatinine, 0.21–0.94 mmol/L), there was no intact IGFBP-3 in urine (20, 25), and urinary IGFBP-3 protease activity was greater than 80%, as measured by the same assay as that used in the present study (25). In the present study even diabetic patients with microalbuminuria and normal renal function had urinary IGFBP-3 protease activities in this range. These findings suggest that the induction of urinary IGFBP-3 proteolysis may be disease specific rather than being related to renal dysfunction per se.
Based on the pattern of inhibition of protease activity by protease inhibitors, the IGFBP-3 protease detected in the urine of five patients with diabetic nephropathy in the present study is a serine protease. Serine protease activity was also observed in patients with renal failure, although three distinct activities with different protease inhibitor profiles were observed in those patients (25).
IGFBP-3 proteolysis is increased in serum from patients with untreated insulin-dependent diabetes mellitus due to the activity of cation-dependent serine proteases (7). Insulin treatment restored protease activity to control levels. Consistent with this observation, levels of intact and proteolyzed IGFBP-3 did not differ in serum from treated diabetic and control subjects in the present study. Further, the presence of diabetic nephropathy did not affect levels of intact IGFBP-3 or its fragments in serum from diabetic patients. This is in contrast to findings in patients with renal failure, who had decreased intact IGFBP-3 levels and increased levels of low molecular mass IGFBP-3 fragments in serum (25).
The source of urinary IGFBP-3 has not been previously defined. Some studies have shown correlations between urinary and serum IGFBP-3 levels and between urinary IGFBP-3 levels and glomerular filtration rate, suggesting that filtration of serum IGFBP-3 may be important (28, 29). IGFBP-3 messenger ribonucleic acid is expressed in human kidney (30), so that local expression may also contribute to urinary IGFBP-3 levels. In patients with renal failure, filtration of serum fragments may contribute to the increased urinary levels of IGFBP-3 fragments. In contrast, the present study suggests that fragmentation of IGFBP-3 occurs within the renal tract in diabetic nephropathy. Firstly, the presence of nephropathy had no effect on levels of IGFBP-3 fragments in serum. Secondly, increased proteolytic activity was detected in urine. We therefore postulate that proteolysis is occurring within the kidney or urinary tract. This may be due to the induction of proteases within the renal tract. In the present study there was a significant correlation between IGFBP-3 proteolysis and urinary albumin excretion. During this process, glomerular leakage of albumin and other proteins is increased; it is possible that this may include filtration of circulating proteases or secretion of proteases from renal tubules into urine.
In diabetic nephropathy, accumulation of mesangial components, such as collagen, laminin, and heparan sulfate proteoglycans, is due to alterations in the balance between protein synthesis and degradation (31). In streptozotocin-induced diabetic rats, matrix metalloproteinase activity in the kidney is decreased (32, 33). In patients with NIDDM, Del Prete et al. has reported down-regulated matrix metalloproteinase-2 and increased tissue inhibitor of matrix metalloproteinase gene expression (34). In diabetic rats, there is decreased albumin degradation at a postglomerular site compared with that in control rats (35). In contrast to the present findings, renal protease activity therefore appears to be generally decreased. This indicates that increased urinary IGFBP-3 proteolysis is not due to a generalized increase in protease activity, but is more likely to be a specific effect.
Proteolysis of IGFBPs has emerged as a possible mechanism by which IGFs are released from high affinity complexes and are subsequently able to bind to IGF-I receptors (5). In the present study, comparison of WIB and WLB clearly shows that the smaller IGFBP-3 fragment has decreased IGF binding affinity, which is consistent with the observations of others (5). A previous study has shown increased urinary, but not plasma, IGF-I levels in patients with microalbuminuria (19). It is possible that the combination of increased IGFBP-3 proteolysis and IGF-I in the urine of patients with diabetic nephropathy results in increased free IGF-I levels. As IGF-I in the glomerular ultrafiltrate of nephrotic rats stimulates proximal tubular collagen secretion (36), increased IGF-I activity may then contribute to the structural changes associated with diabetic nephropathy.
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Acknowledgments |
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Footnotes |
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Received September 10, 1999.
Revised November 23, 1999.
Accepted December 4, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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P < 0.05 vs. control; 