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NDT Advance Access originally published online on March 30, 2006
Nephrology Dialysis Transplantation 2006 21(7):1794-1802; doi:10.1093/ndt/gfl113
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Original Articles: Experimental Nephrology

Fibronectin in blood invokes the development of focal segmental glomerulosclerosis in mouse model

Hao-Ai Shui1, Shuk-Man Ka3, Jung-Chen Lin3, Jien-Huei Lee3, Jong-Shiaw Jin3, Yuh-Feng Lin2, Lai-Fa Sheu3 and Ann Chen3

1 Graduate Institute of Medical Sciences, 2 Department of Internal Medicine and 3 Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC

Correspondence and offprint requests to: Dr Ann Chen, Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, No. 325, Sec. 2, Cheng-Gung Road, Taipei, Taiwan, ROC. Email: doc31717{at}ndmctsgh.edu.tw



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Focal segmental glomerulosclerosis (FSGS) is caused by gradual deposition of extracellular matrix proteins, one of which, fibronectin (FN) is critical for sclerosis development. The origin of the FN deposited at an early stage of FSGS is still unclear.

Methods. For investigating the origin of FN, the onset of increases in FN levels in the serum, glomeruli and urine were studied in a mouse model induced by adriamycin and compared with the time-course of development of glomerulosclerosis and expression of FN mRNA.

Results. In the FSGS mice, serum FN levels were significantly increased as early as the onset of proteinuria on day 4 (7.26±0.37 mg/ml compared with 5.58±0.76 mg/ml in normal controls, P<0.05). This was followed by an increase in glomerular deposition of FN protein on day 7 (FN/actin ratio, 0.216±0.003 compared with 0.039±0.009 in normal controls, P<0.05). Glomerular m-RNA expression was also significantly elevated on day 7, but the locally synthesized FN did not show any increase until day 15. A significant increase in urinary FN protein and focal glomerulosclerosis was seen on day 11.

Conclusions. We infer that FN in blood acts as an initiator of the development of FSGS in this mouse model. In addition, serum and urine FN proteins could serve as useful biomarkers for monitoring the progression of FSGS.

Keywords: adriamycin; biomarker; fibronectin; focal segmental glomerulosclerosis; laser microdissection



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 References
 
The deposition of extracellular matrix (ECM) proteins in glomeruli is a highly dynamic process during the development of focal segmental glomerulosclerosis (FSGS), a chronic glomerular disease which can be caused by environmental toxins, genetic factors, infectious agents, haemodynamic problems or other types of nephritis. The ECM protein deposition in FSGS leads to characteristic histopathological features of sclerotic changes in some glomeruli (focal) and a part of a glomerular capillary tuft (segmental). The ECM protein deposits usually consist of an amorphous substance, made up of precipitated blood proteins, such as fibronectin (FN) and fibrin, as well as other basement membrane or mesangial matrix proteins, such as locally produced FN, laminin and collagen IV [1–8]. These changes result in loss of the structure of the glomerular tuft and finally cause glomerular damage.

Although the mechanism responsible for ECM protein deposition is still inconclusive, it could be caused by matrix deposition exceeding matrix degradation, and many studies have focused on a possible imbalance of in situ synthesis and degradation of ECM proteins in the glomeruli [9,10]. Indeed, some animal experiments have shown that the accumulation of ECM proteins in FSGS is associated with increased ECM protein synthesis coupled with suppression of the expression or activity of proteolytic enzymes, including cathepsins and metalloproteases [10–12]. However, since the glomerular deposits also contain a significant amount of serum FN [6], dynamic changes in serum FN could also affect the development of sclerosis in FSGS.

FN exists as a dimer composed of two nearly identical ~250 kDa subunits linked by a pair of disulfide bonds. FN has many domains responsible for binding to either cell receptors or ECM proteins which are involved in physiological functions, such as cell adhesion, migration, growth and differentiation [13,14]. In pathological conditions, FN could play a pivotal role by acting as a seed for the deposition of ECM proteins around somatic cells, leading to sclerosis or fibrosis of tissue [2,15–17]. The FN deposits could be of either serum or glomerular origin [6,13,18]. Serum-derived FN could be a mediator through which systemic physiological conditions could affect the pathological progress of FSGS. Although FN levels in blood are increased at an early stage in an animal model of kidney damage [4], there is still little information about the contribution of serum FN to early sclerosis in FSGS. The dynamic changes in FN in the serum, glomeruli and urine during the different stages of FSGS remain unclear, hindering an understanding of the origin of the FN and its pathogenic roles in FSGS, especially during the early phase of development of the disease. Taking advantage of an adriamycin-induced FSGS model in mice established in our laboratory [19], we have performed a clinical pathological study over time, and the results strongly suggest that serum FN is implicated in the early development of FSGS.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 References
 
Treatment of animals and analyses of samples
The experiments were performed on 8-week-old female BALB/c mice. The mice were injected intravenously with a single dose of adriamycin (0.1 mg/10 g body weight) or normal saline (control group). Since proteinuria is a typical sign of FSGS and can be used as an indicator of disease progression, a preliminary experiment was performed in which the urinary protein concentration was measured every day in order to choose sampling points, each of which showed a significant change in protein levels compared with the previous day; the days chosen were days 4, 7, 11, 15 and 20. On these days, urine was collected using metabolic cages as described previously [19,20] and blood samples were taken via the retro-orbital venous plexus just before sacrifice; these samples were used to evaluate proteinuria, renal function (creatinine and blood urea nitrogen), and to measure serum and urine FN levels. The mice were then killed and the kidneys examined by microscopy for evidence of sclerosis and by immunohistochemistry (IHC) for FN protein. The glomeruli of the kidneys were isolated using either a laser microdissection (LMD) method for estimation of FN mRNA levels using the real-time polymerase chain reaction (real-time PCR) or a sieving technique to measure FN protein by western blotting.

Determination of urinary protein, serum creatinine and serum urea nitrogen
Samples of blood or urine were microfuged and the serum and clarified urine stored at –70°C until analysed. Proteinuria was measured as the ratio of albumin to creatinine in urine samples as described previously [19]. Creatinine in serum was quantitatively determined using a picric acid colorimetric kit (Sigma 555-1) and serum urea nitrogen was measured using a urease assay kit (Sigma 640–5) [21]. All samples were tested in duplicate.

Measurement of serum and urine FN by ELISA
FN was measured using a human FN competitive enzyme immunoassay kit (Biomedical Technologies Inc., Stoughton, MA) in which the 96-well plates are provided pre-coated with goat anti-rabbit immunoglobulin G and the primary antibody is rabbit antihuman FN. Serum or urine samples (300-fold diluted; 100 µl), were mixed with 50 µl of alkaline phosphatase-conjugated FN (Biomedical Technologies Inc., Stoughton, MA) and 50 µl of primary antibody, then the samples were added to the plates and incubated for 1 h at 37°C. After three washes with PBS-Tween buffer (137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, 0.1% Tween 20, pH 7.4), bound standard FN was then detected using p-nitrophenyl phosphate as chromogen.

Isolation of glomeruli for western blot analysis
The glomeruli were extracted from the kidneys by a sieving technique as described previously [22]. The kidneys were removed, washed with sterile phosphate-buffered saline (PBS) and decapsulated, and the cortex separated from the medulla. A sample of cortex was cut into 1–2 mm3 blocks, which were ground up in PBS and washed sequentially through 60-, 100- and 200-mesh wires using PBS. The glomeruli were collected from the surface of the 200-mesh wire, centrifuged at 750 g for 10 min, and the pellet used for western blot analysis.

Western-blot analysis of total and locally produced FN proteins
The collected glomeruli were lysed by addition of an equal volume of RIPA solution (100 mM Tris, pH 8.0, 300 mM NaCl, 2% Nonidet P-40, 1% sodium deoxycholate, 0.2% SDS) and several cycles of aspiration into, and expulsion from, a pipette, then 12.5 µl of each sample was mixed with equal volume of 2x Laemmili buffer (90 mM Tris-HCl, 10% glycerol, 2% SDS, 100 mM DTT, 0.001% bromophenol blue) and run on a 10% SDS-PAGE gel. The proteins were then electro-blotted onto a nitrocellulose membrane, incubated for 2 h in 20 ml of blocking buffer [Tris-buffered saline, pH 8.0, containing 0.1% Tween 20 (TBST) and 5% skimmed milk], washed three times in TBST and incubated overnight at 4°C with rabbit anti-mouse FN polyclonal antibody (Chemicon; 1/500 dilution in blocking buffer) for non-specifically detecting total FN, or with anti-ECM FN monoclonal antibody (Santa Cruz; 1/200 dilution in blocking buffer) for specific detection of locally produced FN, or with anti-{alpha}-actin antibody (Santa Cruz; 1/500 dilution in blocking buffer) for internal calibration. After three washes with TBST, the blots were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG antibodies (Chemicon) (1/5000 in blocking buffer), washed three times, and the bound antibodies detected by incubation in ECL Plus chemiluminescent reagent (Amersham). Images were captured on X-ray film in a dark room and the protein bands analysed using a gel documentation system (Bio-CAPT). The data are presented as the density of the FN band divided by the density of the {alpha}-actin band.

LMD isolation of glomeruli for real-time PCR
The LMD was performed to precisely isolate glomeruli from kidney sections using a commercialized microdissection system (Microsystems, Wetzlar, Germany), according to the manufacturer's instructions. Frozen sections (10 µm) of renal tissue were cut and mounted on glass slides covered with PEN foil (2.5 µm thick; Leica), as described previously [23]. The sections were then stained by consecutive 30 s incubations (apart from HistoGene: 10 s) in 75% ethanol, DEPC water, HistoGene staining solution (Arcturus, CA, USA), DEPC water, 75% ethanol, 95% ethanol and 100% ethanol. The sections were air-dried for 2 min, then the renal glomeruli were dissected from the frozen sections with the LMD system using a 337 nm nitrogen ultraviolet laser. For each kidney sample, 150 glomeruli were harvested from two consecutive sections, the microdissected glomeruli were collected in microfuge tube cap containing 50 µl of extraction buffer (PicoPure RNA isolation kit, Arcturus, CA, USA), and total RNA was obtained following the manufacturer's instructions. The total RNA was then subjected to real-time PCR analysis as described below.

Measurement of glomerular FN mRNA by real-time PCR
Reverse transcription and real-time PCR was used to measure FN mRNA levels in LMD-isolated glomeruli. For first-strand cDNA synthesis, 3 µg of total RNA was used in a single-round RT reaction in a final volume of 25 µl containing 2.5 µg oligo (dT)12–18 primer, 1 mM dNTPs, 1x first-strand buffer, 0.4 mM DTT, 80 units of RNaseOUT recombinant ribonuclease inhibitor (Invitrogen, CA, USA) and 300 units of superscript II RNase H—reverse transcriptase (Invitrogen, CA, USA).

Real-time PCR was carried out by the iCycler real-time PCR instrument from Bio-Rad, using 10 µl of the RT reaction mixture, 500 nM gene-specific primers and 12.5 µl Bio-Rad iQ SYBR Green supermix in a total volume of 25 µl. The gene-specific primers are designed to evaluate the expression of either mouse FN (Forward primer: 5'-ACAGAAATGACCATTGAAGG-3'; Reverse primer: 5'-TGTCTGGAGAAAGGTTGATT-3') or GAPDH (Forward primer: 5'-TCCGCCCCTTCTGCCGATG-3'; Reverse primer: 5'-CACGGAAGGCCATGCCAGTGA-3'). The FN and GAPDH cDNA were amplified simultaneously by denaturation at 94°C for 2 min, followed by 40 cycles of 94°C for 15 s, 55°C for 30 s and 72°C for 30 s, and a final extension at 72°C for 10 min. The FN mRNA levels were normalized to GAPDH mRNA levels by a comparative threshold cycle (2–[delta]Ct) method which converts differences of cycle numbers between FN and GAPDH to FN/GAPDH ratios.

Histopathology and Immunohistochemistry
Renal tissues were fixed in 10% buffered formalin for routine histopathological evaluation. The paraffin-fixed renal sections were dehydrated in graded ethanol, embedded in paraffin, stained with haematoxylin and eosin (HE), and examined using an optical light microscope (Olympus, Tokoyo, Japan).

Severity of sclerosis was semi-quantified by morphological changes on a scale of 1–4, according to a previous method [19]. Briefly, a score of 1 was equivalent to 25% of the glomerulus showing sclerosis and a score of 4 to involvement of 100% of the glomerulus. The total score was obtained by addition of the individual scores for 50 randomly selected glomeruli in a section.

For IHC, renal tissues were embedded in OCT-medium (Optimal Cutting Medium) and cut into 5 µm frozen sections. After drying for at least 30 min at room temperature, the sections were immersed in acetone for 5 min, then air-dried. Bovine serum albumin dissolved in TBST was used for blocking and TBST was used for all washing steps. After treatment with anti-human FN antibody (Chemicon; 1/200 dilution) and washing with TBST, horseradish peroxidase-conjugated protein G (Pierce, IL, USA) was applied to the sections for 1 h as described previously [19]. Reaction products were visualized using 3,3'-diaminobenzidine chromogen (DAKO, Carpinteria, CA, USA), and the slides were counterstained lightly with haematoxylin.

A semi-quantitative evaluation of glomerular staining for FN was performed using a previously described method [19]. Briefly, 50 randomly selected glomeruli were examined in each section and assigned values of staining intensity from 0 to 3. A total score was calculated for each specimen according to the following equation: Total intensity score = (% of negative intensity glomeruli x 0) + (% of trace intensity glomeruli x 0.5) + (% of intensity 1 + glomeruli x 1) + (% of intensity 2 + glomeruli x 2) + (% of intensity 3 + glomeruli x 3). The values ranged from 0 to a maximum of 300.

Statistics
The data are presented as the mean±SE. Student's t-test was used for the statistical analysis. Differences were considered significant at P<0.05.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 References
 
Early-appearing proteinuria and late-occurring renal failure in FSGS
To confirm the establishment of the FSGS model, proteinuria was evaluated in the adriamycin-treated mice. As shown in Figure 1A, the adriamycin-treated mice began to show significant proteinuria (albumin/creatinine ratio, 0.86±0.04) compared with normal control mice (0.25±0.04) (P<0.05) on day 4, then urinary protein levels remained high throughout the experiment. Serum levels of urea nitrogen and creatinine, two parameters for evaluating renal function, were also examined, and showed a significant and persistent increase from day 11 (47.6±2.7 and 0.65±0.07 mg/dl compared with 23.9±2.8 and 0.48±0.04 mg/dl in normal controls, respectively, P<0.05) (Figure 1B and C), indicating a late-onset and ongoing deterioration of renal function in the FSGS model.


Figure 1
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  		Fig. 1. Clinical manifestation of adriamycin-induced FSGS in mice. Three typical clinical parameters for evaluating renal function, (A) urinary protein, (B) blood urea nitrogen and (C) serum creatinine levels, were monitored over time. Urine and blood samples were taken at the indicated time after adriamycin treatment. The data are shown as the mean±SE. *Significant difference compared with the basal level, n = 6, P<0.05.

 
Dynamic changes in FN levels in serum and urine
Serum and urine FN levels at different stages of FSGS are shown in Figure 2. Compared with basal serum FN levels (5.58±0.76 mg/ml), serum FN levels showed a significant increase as early as day 4 (7.26±0.37 mg/ml) after adriamycin injection and increased continuously throughout the experiment (day 7: 7.853±0.606; day 11: 11.121±0.103; day 15: 10.96±0.95; day 20: 11.51±0.68 mg/ml; P<0.05 for each) (Figure 2A). Compared with the basal level of 0.007±0.002, urinary levels of FN corrected for urine creatinine were dramatically increased (P<0.05) on day 11 (0.028±0.005) and plateaued on day 20 (0.058±0.00008) (Figure 2B).


Figure 2
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  		Fig. 2. Time-course of changes in serum and urinary FN levels. Protein concentrations of FN measured by ELISA in the (A) serum and (B) urine are shown for the adriamycin-treated animals (solid line) and the control animals (dashed line).The urine data are normalized by dividing the FN levels by the corresponding concentration of urinary creatinine. *Significant differences in protein levels compared with the basal level, n = 6, P<0.05.

 
Glomerular FN protein and mRNA levels at various time points
The western blot results for total and locally synthesized FN in the isolated glomeruli from FSGS mice are shown in Figure 3A and B, respectively; semi-quantitative data of the FN expressions normalized by actin levels are depicted in Figure 3C. Glomerular FN mRNA levels at different time-points in the FSGS model were measured by real-time PCR and depicted in Figure 3D.


Figure 3
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  		Fig. 3. Analysis of glomerular FN protein and mRNA levels. (A) Typical western blot showing the time-course of changes in total FN and actin protein levels after adriamycin treatment. (B) Western blot showing dynamic changes of ECM FN and actin protein levels. (C) Bar chart showing the FN/actin ratios of both total and ECM FNs. (D) Bar chart showing the FN/GAPDH ratios derived from real-time PCR quantification of glomerular mRNA at different times after adriamycin treatment. The data are shown as the mean±SE. *Significant difference from the basal value (day 1), n = 6, P<0.05.

 
Unlike the unchanged levels of locally synthesized FN proteins on day 7 (FN/actin ratio, 0.0027±0.0007) as compared with basal levels (FN/actin ratio, 0.0033±0.0009) (P>0.05) (grey bars of Figure 3C), glomerular FN mRNA and total FN protein increased significantly at the same time in comparison with their controls (0.00026±0.00006 and 0.216±0.03 compared with 0.00009±0.00006 and 0.039±0.005 in FN/GAPDH and FN/actin ratios, respectively, P<0.05) (Figure 3D and black bars of Figure 3C), indicating that, at an early stage of FSGS, enhanced expression of FN gene (Figure 3D) does not lead to accumulation of ECM FN proteins (Figure 3B) and cannot account for the increased FN deposition in glomeruli (Figure 3A).

Western blot analysis of glomerular FN fragmentation
For elucidating the protein metabolism of FN in glomeruli, the entire range of molecular weight of western blot from Figure 3A is shown in Figure 4. The FN fragments (<250 kDa) became more and more abundant accompanied by the increased deposition of full-length FN (250 kDa) from day 7 to day 15, and then diminished on day 20 when the full-length FN remained at a high level (Figures 3A, C and 4). The dynamic changes of full-lengh FN and its fragments strongly indicate that rate of FN catabolism does not decrease when full-length FN protein begins to accumulate, ruling out a possibility that FN deposition at an early FSGS stage is caused by reduced catabolism.


Figure 4
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  		Fig. 4. Western blot analysis of glomerular FN fragmentation. Time points for sample collections after adriamycin treatment are indicated on the top of the gel. Molecular weight markers are shown on the left of the gel. The position for full-length FN and distribution for FN fragments are depicted on the right site.

 
Glomerulosclerosis and FN deposition
To evaluate glomerulosclerosis and FN deposition in adriamycin-treated mice, histopathology and IHC were performed on kidney sections. As shown in Figure 5, adriamycin-induced typical glomerulosclerosis during the late stage (day 20) of FSGS (sclerosis index of 69.9±7.3 compared with 0 in controls; Figure 5C). The IHC also showed a striking deposition of FN in the sclerotic area of the glomeruli at this stage (Figure 6A) compared with the small amount of FN protein seen in normal glomeruli (Figure 6B), the IHC scores being 275±22 in the FSGS animals and 80±10 in controls (P<0.05). The FN deposition in the glomeruli correlated well with the development of glomerulosclerosis.


Figure 5
Figure 5
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  		Fig. 5. Histopathology of the kidneys in adriamycin-treated mice. (A) HE staining of the kidneys in treated mice shows severe sclerosis of the glomeruli with amorphous ECM protein deposition, while (B) control mice display a normal glomerular structure. (C) The sclerosis score calculated as described in the subjects and methods section, n = 6, *P<0.05, compared with day 1.

 

Figure 6
Figure 6
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  		Fig. 6. IHC showing glomerular FN deposition. (A) Adriamycin-treated mice have glomeruli with massive FN deposition, whereas (B) control mice show only very low FN levels. (C) Scores for FN deposition in the glomeruli at different stages of FSGS, n = 6, *P<0.05, compared with day 1.

 
Combined analysis of FN levels in serum, glomeruli and urine
The time of onset of the increase in FN levels in the serum, glomeruli and urine can provide valuable information regarding cause–effect relationships between serum-borne, glomerulus-produced and urine-secreted FN protein. The dynamic changes in FN levels in all three compartments are shown in Figure 7; in this figure, the basal values for the FN levels in the different compartments are set to one and the FN levels at different time points are expressed as the test point/basal ratio. As shown in Figure 7, the earliest significant increase in FN protein levels was seen in the serum (day 4), then in the glomeruli (day 7, total FN protein) and finally in the urine (day 11), implicating that the elevated serum FN might contribute to the following FN deposition in glomeruli and appearance in urine. Although FN mRNA was elevated as early as FN deposits in the glomeruli (day 7), its local product, the ECM FN, was not increased until day 15, indicating dissociation between FN gene transcription and protein metabolism (mRNA translation and protein degradation).


Figure 7
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  		Fig. 7. Normalized FN levels in serum, glomeruli and urine. The results for the different time points in each compartment were normalized to the corresponding basal level (day 1 sample).

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
References
 
Regardless of whether serum FN levels are increased or not, they are always high enough in humans (300 µg/ml) to contribute to the deposition of FN in glomeruli [13]. However, the increase in serum FN levels seen in the present study (Figures 2 and 7) might accelerate deposition and exacerbate FSGS. After leaking from the blood to the basement membrane and mesangial area, FN can bind to various ECM proteins and cell receptors (integrin) [13,14]. As a result, blood FN can be deposited until all binding sites in the glomeruli are saturated. Our results show that serum FN levels were increased slightly, but significantly, on day 4 (Figures 2 and 7), earlier than glomerular FN deposition on day 7 (Figures 3A, C and 7); this increase must be partly responsible for the early glomerular deposition, though local FN gene expression was also increased at this time (Figures 3D and 7). Similar findings of a very early increase in plasma FN levels have been seen in animals with immune and toxic damage of the kidney, although, unlike in our study, tissue deposition of FN protein and FN mRNA levels were not simultaneously analysed [4]. In addition, altered plasma FN levels have been reported in many disorders, such as, liver cancer and infectious diseases, and might be suitable as a non-specific biomarker to improve diagnosis and prognosis in combination with other conventional methods [24–26]. Accordingly, although the mechanism is still unclear, the increase in serum FN levels seen in our study has potential as an indicator for improving the early diagnosis of FSGS. Since the liver is the major source of serum FN under normal conditions [13], the increase in serum FN levels might arise from adriamycin-enhanced output of FN from the liver. This needs to be confirmed in human FSGS, especially for FSGS induced by certain factors that could also affect liver function.

Previous reports have focused on the role of up-regulation of FN gene transcription in FSGS without simultaneously correlating transcription with FN protein levels in the glomeruli, especially during the very early stage of FSGS [27–30]. However, by concurrently monitoring FN mRNA and protein levels (Figure 3) during the early stage, our results strongly indicate that the early marked deposition of FN protein (Figure 3A and C) cannot be easily explained by the simultaneously increased FN mRNA levels (Figure 3D), because local ECM FN protein, which is translated from mRNA, does not show any comparable accumulation (Figure 3B and C). This is the first report showing that the FN deposition during the early stage of FSGS is not caused by increased gene transcription or mRNA translation. A similar result was obtained in immune-mediated nephritis models with different pathogeneses from FSGS, showing that, at the early time point, the increased glomerular FN comes predominantly from the plasma rather than the gene expression [6,31]. However, other possibilities which involve changes in FN metabolism, such as reduced protein degradation, could lead to the same outcome and were suggested to be considered [6,31]. Since our results clearly demonstrate that FN protein degradation does not decrease in early FSGS in glomeruli (Figure 4), this possibility can be ruled out. The pathogenic factors influencing the balance between blood deposition and local synthesis of FN are still unclear, and could be cytokines affecting integrin and FN expressions, such as IL-4 and TGF-ß, respectively [7].

Various biochemical substances in urine have been suggested as non-invasive diagnostic markers for urogenital diseases, such as urine albumin for nephritis and some specific urine markers for prostate and bladder cancer [32–34]. Urine biomarkers are more valuable for monitoring severity of disease than blood biomarkers, as urine sampling is a non-invasive procedure. In contrast to serum FN levels, which, as discussed in a previous paragraph, could act as a non-specific biomarker for evaluating the predisposition of FSGS, urinary FN levels, which increase after glomerular FN deposition, could serve as an indicator of the severity of FSGS, as the increase in the urine FN/creatinine ratio correlated well with the observed extent of sclerosis and loss of renal function (Figure 2B compared with Figures 1B, C and 5). If a similar correlation were seen in humans, urinary FN levels could be a valuable marker for prognosis of FSGS in clinics. The reason for the increase in urinary FN levels is still unclear, but might be the increased leakage of FN due to many factors, including a breakdown of the filtration barrier, a sclerosis-induced reduction in FN binding sites, and decreased FN degradation and re-absorption [8].

In summary, our data support the idea that the early glomerular deposition of FN in FSGS is caused by FN originating in the blood, rather than the glomerulus, and could play a pathogenic role in initiating the later development of glomerulosclerosis. Importantly, both serum and urinary FN proteins might serve as useful biomarkers for assisting in diagnosis and in the monitoring of FSGS.



   Acknowledgments
 
This study was supported by grants from the National Science Council (NSC93-2320-B-016-026; NSC 93-2314-B-016-066), Taiwan, ROC.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 19.12.05
Accepted in revised form: 22. 2.06


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