Immunoelectron microscopic study on type I, II and III TGF-ß receptors on visceral glomerular epithelial cells in relation to glomerular basement membrane alterations in proteinuric rats

Yoshihide Fujigaki1, Takuya Watanabe1, Naoki Ikegaya1, Katsuhiko Yonemura1, Di Fei Sun1, Akira Hishida1, Tatsuo Yamamoto2, Kenichiro Kojima3 and Mitsumasa Nagase3

1 First Department of Medicine, Hamamatsu University School of Medicine, 2 Division of Nephrology, Seirei Hamamatsu General Hospital, Hamamatsu and 3 Department of Medicine, Teikyo University School of Medicine, Itabashi, Tokyo, Japan

Correspondence and offprint requests to: Yoshihide Fujigaki, MD, First Department of Medicine, Hamamatsu University School of Medicine, Handa-cho 3600, 431–3192 Hamamatsu, Japan.



   Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. Transforming growth factor (TGF)-ß is a regulator of extracellular matrix accumulation. Both TGF-ß receptors, type I (TßRI) and type II (TßRII), may be required for signal transduction in the TGF-ß pathway. The aim of this study was to investigate the relationship between the TGF-ß pathways and glomerular basement membrane (GBM) accumulation in vivo.

Methods. We examined TßRI, II, and III protein expression on visceral glomerular epithelial cells (GEP) in relation to GBM alterations in passive Heymann nephritis (PHN), anti-GBM nephritis and anti-thymocyte serum (ATS) nephritis. Renal tissues were examined by pre-embedding immunoelectron microscopy 3, 7 and 14 days after induction of nephritis in rats.

Results. In normal control rats TßRI was not detected on GEP, TßRII expression was very occasionally found on GEP and TßRIII was seen in the cytoplasm of the GEP. TßRI, TßRII, and TßRIII were constitutively expressed on glomerular endothelial cells. By day 3 of anti-GBM nephritis and PHN, expression of TßRI, TßRII, and TßRIII was still similar to that of normal control rats, and GBM alterations in both models were not prominent except for deposit formation in PHN. From day 7 onwards, in both models, expression of TßRI and TßRII on GEP increased in association with GBM thickening. Expression of TßRIII in the cytoplasm of the GEP was increased, with occasional positive staining being seen on the urinary surface of the GEP from day 7 onwards. On the other hand, at day 3 of ATS nephritis, increased expression of TßRI and TßRII on GEP was noted, but from day 7 onwards, expression of TßR II on GEP dramatically decreased. Expression of TßRIII in the cytoplasm of the GEP also transiently increased at day 3. GBM thickening was not noted in ATS nephritis.

Conclusions. The results suggest that persistent upregulation of expression of TßRI, TßRII and possibly TßRIII on GEP may contribute to GBM matrix accumulation in vivo.

Keywords: glomerular basement membrane; glomerular epithelial cell; glomerulonephritis; TGF-ß; TGF-ß receptor



   Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Transforming growth factor-ß (TGF-ß) is a multipotent regulator of cell proliferation and extracellular matrix (ECM) accumulation. TGF-ß functions by stimulating matrix synthesis, increasing protease inhibitors, and altering integrins (matrix receptors) to allow increased binding and depostition of ECM components [1,2]. The activation of TGF-ß is mediated through binding to cell surface receptors [3]. Three major types of TGF-ß receptors (TßR), type I (TßRI), type II (TßRII), and type III (TßRIII) have been identified in most cells [4,5]. These receptors form heteromeric receptor complexes in ligand binding. Both TßRI and TßRII may be required for signal transduction in the TGF-ß pathway [6,7], whereas TßRIII has been shown to present or deliver TGF-ß to the signalling receptors [8,9] and thus enhance TGF-ß activity [10].

The glomerular basement membrane (GBM) is also an ECM and derives its matrix material mainly from the visceral glomerular epithelial cell (GEP) in the mature glomerulus [11]. However, little is known about the role of the TGF-ß pathway in accumulation of the GBM, mainly because there is an ongoing debate concerning the origin of cultured GEP cell lines [1214]. Nakamura et al. reported that TGF-ß1 increased the production of components of the GBM (fibronectin and type IV collagen) by rat GEP in their particular culture system [15]. Recently Shankland et al. reported the functional consequences of increased expression and activity of TGF-ß2 and TGF-ß3, as well as TßRI and TßRII on the GEP, may be linked to the expansion of GBM in passive Heymann nephritis (PHN), a rat model of membranous nephropathy [16]. These data suggest that the TGF-ß pathway may contribute to GBM accumulation, but that the various TGF-ß isoforms might exert their function on the GEP in respect of GBM accumulation in different ways.

The aim of this study was to investigate the relationship between the TGF-ß pathways and GBM accumulation in vivo. As both TßRI and TßRII cooperate for TGF-ß signal transduction [6,7], they are both apparently required to be present on a particular cell type for TGF-ß function to ensue. Thus expression of TßRI, TßRII, and TßRIII proteins on the GEP at the ultrastructural level was examined in three rat models of nephritis in relation to GBM thickening. For this purpose PHN (complement mediated GEP injury) [17], anti-GBM nephritis (complement-independent immunologic injury) [16], and anti-thymocyte serum (ATS) nephritis (complement-mediated immunologic model of mesangial cell injury) [18] were selected.



   Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antibodies
Anti-Fx1A (antigen on the brush border of proximal tubules) antiserum, anti-rat GBM antiserum and anti-rat thymocyte antiserum were prepared in a sheep according to previous reports [2,19,20]. The globulin fractions of anti-Fx1A antiserum and anti-rat GBM antiserum were obtained by ammonium-sulfate precipitation, yielding IgG concentrations of 60 and 30 mg/ml in 0.01 M phosphate buffered saline (PBS) respectively. Rabbit anti-human TßRI, TßRII, and TßRIII antibodies were directed against the following synthetic peptides. Amino acid residues: 158–179 of activin receptor-like kinase 5 (ALK-5); 245–266 of TßRII; and 819–830 of TßRIII (intracellular domain) respectively (kindly provided by Dr Kohei Miyazono, The Cancer Institute, Tokyo, Japan) [2123].

Animals and estimation of proteinuria
Male Wistar rats (150–200 g) (Japan SLC Co., Shizuoka, Japan) were used. Twenty-four-hour urinary specimens were collected in metabolic cages and the protein content was measured by the biuret method [24].

Induction of experimental nephritis
PHN, anti-GBM nephritis and ATS nephritis were induced by i.v. injection of sheep anti-FX1A IgG (0.5 ml in PBS), sheep anti-rat GBM IgG (1.0 ml in PBS) or sheep anti-thymocyte antiserum (0.5 ml) respectively. Groups of three rats were sacrificed 3, 7 and 14 days after disease induction, four normal rats served as controls.

Histologic examination
To perform light microscopic examination, renal tissues were fixed in 4% paraformaldehyde overnight at room temperature. Paraffin sections were cut at 3 µm and the sections were stained with periodic acid-Schiff reagent.

Immunoelectron microscopy for detection of TßR
In preliminary studies localization of the antigen recognized by the TGF-ß receptor antibodies used could not be satisfactorily visualized using a post-embedding immunogold technique with Unicryl resins (British BioCell, UK). Therefore pre-embedding immunoelectron microscopy using 4% paraformaldehyde-fixed and paraffin-embedded sections was adopted.

After deparaffinization, endogenous peroxidase was blocked in 3% hydrogen peroxide for 30 min. Sections were then incubated with 10% normal donkey serum for 20 min, followed by incubation with the primary antibodies (TßRI–III) appropriately diluted in PBS (pH 7.4) containing 3% normal donkey serum for 60 min. Following washes in PBS, the sections were incubated with biotinylated donkey anti-rabbit IgG (Fab')2 (Chemicon International Inc., Temecula, CA) for 60 min, washed in PBS, then incubated with streptavidin-conjugated peroxidase for 30 min. After the sections had been fixed in 2% glutaraldehyde for 10 min, reaction products were visualized by incubating with diaminobenzidine, then the sections were postfixed in 1% osmium tetroxide for 2 h, dehydrated in a graded ethanol series, and embedded in Epon 812. All incubations were performed at room temperature. Ultra-thin sections were stained with lead citrate for 1.5 min and examined with a JEM-1220 electron microscope (JEOL, Tokyo, Japan). Histological controls were obtained by omitting the primary antibodies or replacing the primary antibodies with normal rabbit IgG at equivalent concentrations. For the semi-quantitative assessment of TßRI and TßRII immunostaining on GEP, a minimum of eight electron micrographs of the peripheral capillary wall sections were taken for each glomerulum at a magnification of x20000. At least three glomeruli per rat were examined. Staining intensity on prints was graded using a scale ranging from - (negative) to ++ (strongly positive) with two intermediate staining intensities, viz. ±(weakly positive) and + (positive). When the immunostaining showed focal distribution, the individual length of the GBM stretches with positive immunostaining on the surface of GEP, was measured. Then the total length of the stretches, as a percentage of whole capillary loop length per animal, was calculated. To minimize observation bias, staining intensity was judged by two investigators without knowledge of the group from which the prints were obtained.

Measurement of the GBM thickness
The ultra-thin sections not subjected to immunostaining were stained with uranyl acetate and lead citrate. A minimum of eight electron micrographs of sections of the peripheral capillary wall were taken for each glomerulum at a magnification of x20 000 and prints were made at a magnification of x40 000. At least two glomeruli per rat were examined. In anti-GBM nephritis, regions of the capillary wall with apparent subendothelial widening were excluded when measuring GBM thickness. In these micrographs, the distance between the endothelial and epithelial plasma membranes were measured at 1.0-µm intervals. The thickness of the GBM was expressed as the distance between the epithelial and the endothelial plasma membranes, using a computerized image analyser (MPO-Videoplan, Carl Zeiss Inc., Germany).



   Results
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 Introduction
 Methods
 Results
 Discussion
 References
 
Profiles of experimental nephritis
Before induction of nephritis no abnormal urinary protein excretion (<10 mg/day) was observed in any rat in any group. In PHN, abnormal urinary protein excretion was seen at day 7 (138.2±12.0 mg/day, mean±SD) which had increased by day 14 (211.6±10.3 mg/day). In anti-GBM nephritis proteinuria could be found at day 3 (139.4±18.0 mg/day), increasing further thereafter (day 7, 151.7±7.6 mg/day; day 14, 222.4±7.4 mg/day). In ATS nephritis, abnormal urinary protein excretion could be seen at day 3 (342.1±40.5 mg/day), keeping to the same level until day 7 (307.1± 19.8 mg/day), followed by a reduction by day 14 (35.4±6.1 mg/day).

The histologic profiles of the three nephritis models used in this study (Figure 1Go) were in line with those previously reported [16,17]. Briefly, PHN showed diffuse, slight glomerular capillary wall thickening, without mesangial hypercellurality, at 7 days onwards (Figure 1BGo). Anti-GBM nephritis showed increased glomerular cellularity with mesangial matrix expansion by day 3, and proliferation of endothelial and mesangial cells with double contouring and segmental thickening of the glomerular capillary wall at 7 days onwards (Figure 1CGo). ATS nephritis showed mesangiolysis and segmental microaneurysmal ballooning at day 3 (Figure 1DGo), and mesangial cell proliferation with mesangial matrix expansion at day 7 (Figure 1EGo). Mesangial cell proliferation and mesangial matrix expansion tended to subside slightly by day 14 (Figure 1FGo). During the disease course of ATS nephritis no apparent capillary wall thickening could be seen (Figures 1D–FGo).



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Fig. 1. Light micrographs of glomeruli from rat kidneys in (A) normal rats, (B) PHN at 14 days, (C) anti-GBM nephritis at 14 days, (D) ATS nephritis at 3 days, (E) ATS nephritis at 7 days and (F) ATS nephritis at 14 days. (PAS staining).

 
Ultrastructural alterations and TßR protein expression in the glomerular capillary wall
A summary of the results of TßRI and TßRII immunostaining on the GEP is shown in Table 1Go.


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Table 1. Summary of results of TßRI and TßRII immunostaining on glomerular epithelial cells
 
Normal rat
In normal control rats, positive immunostaining for TßRI was not seen in GEP (Figure 2AGo). TßRII immunostaining was found very occasionally on the urinary surface of the GEP (Figure 2BGo) and TßRIII immunostainig was seen in the cytoplasm of the GEP (Figure 6AGo). All three TßR were constitutively expressed on the glomerular endothelial cells (GEN) (Figures 2A, B and 6AGoGo), but immunostaining for TßRIII was faint (Figure 6AGo). In histologic control sections (either omitting the first antibodies or replacing the first antibody by normal rabbit serum) no specific staining was found (Figure 2CGo).



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Fig. 2. Immunoelectron micrographs of glomerular capillary walls stained by TßRI (A) and TßRII (B) in normal rats. (C) Histologic control, replacement of the primary antibodies with normal rabbit IgG. P, podocyte; CL, capillary lumen. Bar 0.5 µm.

 


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Fig. 6. Immunoelectron micrographs of glomerular capillary walls stained by TßRIII in normal rats (A) PHN at 7 days (B), anti-GBM nephritis at 7 days (C), and ATS nephritis at 3 days (D). P, podocyte; CL, capillary lumen; US, urinary space. Bar 1.0 µm.

 
PHN
By day 3 of PHN, the expression of all three TßR was still similar to that observed in normal control rats (Figures 3A and DGo). At day 7 onwards, expression of TßRI and TßRII on the urinary surface of GEP diffusely increased in association with GBM thickening (Figures 3B, C, E and FGo). Expression of TßRIII in the cytoplasm of the GEP was also increased, with occasional staining on the urinary surface of the GEP at day 7 onwards (Figure 6BGo). Expression of all three TßR on the GEN did not change during the disease course (Figures 3 and 6BGoGo).



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Fig. 3. Immunoelectron micrographs of glomerular capillary walls stained by TßRI (AC) and TßRII (DF) in PHN at 3 days (A,D), at 7 days (B,E), and at 14 days (C,F). P, podocyte; CL, capillary lumen; asterisks, immune deposits. Bar 0.5 µm.

 
Anti-GBM nephritis
By day 3 of anti-GBM nephritis, immunostaining for all three TßR was still similar to that of normal control rats (Figures 4A and DGo) but with focal GEN detachment from the GBM (Figure 4AGo). At day 7 onwards, segmental immunostaining for TßRI and TßRII on the urinary surface of the GEP was found, and the capillary walls showed segmental GBM thickening with irregularity of the endothelial side of the GBM (Figures 4B, C, E and FGo). Expression of TßRIII in the cytoplasm of the GEP was also increased, with occasional staining on the urinary surface of the GEP at day 7 onwards (Figure 6CGo). Increased TßRI and TßRII on the GEN and increased TßRIII in the cytoplasm of the swollen GEN was also found at day 7 onwards (Figures 4 and 6CGoGo).



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Fig. 4. Immunoelectron micrographs of glomerular capillary walls stained by TßRI (AC) and TßRII (DF) in anti-GBM nephritis at 3 days (A,D), at 7 days (B,E), and at 14 days (C,F). P, podocyte; CL, capillary lumen; asterisks, immune deposits. Bar 0.5 µm.

 
ATS nephritis
At day 3 of ATS nephritis, increased diffuse immunostaining for TßRI and TßRII on the urinary surface of the GEP was noted (Figures 5A and DGo). At day 7 immunostaining for TßRII on the GEP dramatically decreased (Figure 5EGo) but immunostaining for TßRI was still increased (Figure 5BGo). At day 14 staining for TßRI and TßR1II returned to normal levels (Figures 5C and FGo). Immunostaining for TßRIII in the cytoplasm of the GEP transiently increased at day 3 (Figure 6DGo). Immunostaining for all three TßR on the GEN was also transiently increased at day 3 (Figures 5A, D and 6DGoGo). The GBM seemed to be stretched transiently at day 3 (Figure 5Go).



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Fig. 5. Immunoelectron micrographs of glomerular capillary walls stained by TßRI (AC) and TßRII (DF) in ATS nephritis at 3 days (A,D), at 7 days (B,E), and at 14 days (C,F). P, podocyte; CL, capillary lumen. Bar 0.5 µm.

 
Morphometric analysis of the GBM thickness
Results of the measurement of GBM thickness in normal control rats and in the three nephritis models at days 3, 7 and 14 are shown in Figure 7Go. In both PHN nephritis and anti-GBM nephritis, at days 7 and 14, mean thickness of the GBM was significantly higher than in normal control rats. In ATS nephritis the mean thickness of the GBM did not increase compared with normal control rats. On the contrary, GBM thickness at day 3 of ATS nephritis was significantly lower than in normal control rats.



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Fig. 7. GBM thickness in (A) normal rats, (B) PHN, (C) anti-GBM nephritis, and (D) ATS nephritis. Data are expressed as means±SD nanometers from mean values per glomerulus. 3d, 3 days; 7d, 7 days; 14d, 14 days; N, number of glomeruli examined. *P<0.05 vs normal rats, **P<0.001, #P<0.05, ##P<0.001 (ANOVA followed by Scheffe's F test).

 


   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we examined, at the ultrastructural level, the relationship between GBM alterations and the TGF-ß pathway, as judged by expression of TßR protein on the GEP and GEN in three rat models of nephrtitis. The thickening of the GBM increased over time in PHN and anti-GBM nephritis, and correlated with the increase in the expression of TßRI, TßRII, and TßRIII. The GBM thickness in ATS rats did not change essentially over time but TßRI, TßRII, and TßRIII were upregulated at day 3 after insult and decreased thereafter. These data showed that the GBM thickening was closely associated with the persistent expression of TßRI and TßRII, and possibly TßRIII on the GEP. Thus it is suggested that the persistent expression of TßR on the GEP may contribute to GBM accumulation.

The causality of TßR upregulation contributing to the GBM thickening in PHN and anti-GBM nephritis is not clear. However, Nakamura et al. firstly reported that TGF-ß1 increased the production of components of the GBM (fibronectin and type IV collagen) by rat GEP in vitro [15]. Shankland et al. reported that the functional consequences of increased expression and activity of TGF-ß2 and TGF-ß3, as well as TßRI and TßRII, on the GEP in vivo may be linked to the expansion of GBM in PHN [16]. It is well known that GBM thickening is a prominent feature of diabetic nephropathy, and van Det et al. reported that high glucose concentrations stimulated the synthesis of fibronectin in human GEP in vitro and that this effect was mediated by induction of TGF-ß [25]. Recently, Isaka et al. reported that TßRII fused to IgG-Fc portion gene transfection inhibited the biological activities of TGF-ß in the rat diabetic kidney, and blocked GBM thickening [26]. These data suggest that TGF-ß pathway may contribute to GBM accumulation through TßR upregulation on the GEP.

In this study expression of TGF-ß was not examined. However, protein expression and/or mRNA levels for TGF-ß in glomeruli and/or kidney cortex were reported to increase in these three nephritis models [16,2729], and the kinetics of TGF-ß expression in the glomeruli roughly paralleled that of immunostaining for TßR on the GEP in the present study. Besides TGF-ß production by the GEP, the source of the TGF-ß could be either the proliferating mesangial cells and/or the macrophages in both anti-GBM nephritis and ATS nephritis. The increase in the number of mesangial and inflammatory cells reported [2,17,27,30] also roughly paralleled TßR expression on the GEP.

The stimulus for TßR expression on the GEP remains to be determined since the degree of proteinuria seemed unrelated to the TßR expression in these three nephritis models. In PHN it is conceivable that immunologic GEP injury [17] is the stimulus for TßR expression. Recently, Sanker et al. have reported that changes in the matrix environment can modulate expression of TßR and alter cell responsiveness to TGF-ß1 in cultured microvascular endothelial cells [31]. Therefore altered GBM matrix, caused by deposit formation in PHN and/or binding of anti-GBM antibody to the GBM in anti-GBM nephritis [17] as well as the infiltrated macrophages in anti-GBM nephritis [2,27] and ATS nephritis [30], might stimulate TßR upregulation on the GEP. In ATS nephritis the microaneurysmal ballooning and decrease in the GBM thickness at day 3 of ATS nephritis seen in this study indicate the magnitude of the stretching forces acting of mesangial cells and the capillary wall. Thus, it is possible that mechanical stretching might transiently induce the upregulation of TGF-ß and TßR on the GEP in ATS nephritis because mechanical stretching could induce expression of TGF-ß and ECM components in cultured rat mesangial cells [32].

The roles of TßR found on the GEN in GBM thickening have not been evaluated. In this study, the intensity of staining for all three TßR on the GEN, in anti-GBM nephritis from day 7, seemed to be increased in association with GBM thickening. However, GBM newly formed at the endothelial side could not be clearly seen in anti-GBM nephritis until day 14. In PHN, changes in TßR on the GEN were not seen at days 7 and 14, when significant GBM thickening was observed. These findings suggest that it is less likely that GBM thickening in glomerulonephritis is generally mediated by an effect of the TGF-ß pathway on the GEN. However, a contribution of the GEN to GBM thickening could not be excluded in anti-GBM nephritis because it was reported that TGF-ß1 increased the production of components of the GBM (proteoglycans) by cultured bovine GEN [33].

Both TßRI and TßII may be required for signal transduction in the TGF-ß pathway [6,7], and TßRIII may enhance TGF-ß activity [810]. As the presence of TßR partially determines cellular responsiveness to TGF-ß [31], increased expression of the three TßR suggests increased cellular sensitivity to TGF-ß. We conclude that persistent upregulation of expression of TßRI, TßRII and possibly TßRIII on GEP may contribute to pathological matrix accumulation (GBM matrix accumulation) through TGF-ß pathways in vivo.



   Acknowledgments
 
The authors wish to thank Mr Isao Ohta, Central Laboratory for Ultrastructure Resrch, Hamamstsu University School of Medicine, Hamamatsu, Japan for excellent technical assistance. This work was supported by research grant (No. 09770844) sponsored by The Ministry of Education Science, Sports and Culture in Japan, and partially presented at XXXVth congress of the ERA-EDTA in Rimini, Italy, 1998.



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 Abstract
 Introduction
 Methods
 Results
 Discussion
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Received for publication: 14. 5.99
Accepted in revised form: 31. 8.99