1Department of Pathology and 2Department of Nephrology, University Medical Center Nijmegen, Nijmegen, The Netherlands
Correspondence and offprint requests to: B. Smeets, Department of Pathology, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Email: b.smeets{at}pathol.umcn.nl
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Abstract |
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Methods. Albuminuria was measured in Thy-1.1 transgenic mice after injection of different doses of anti-Thy-1.1 mAb and at different time points within the first 24 h after injection. Podocytic foot processes and slit pore diameter were quantitated by electron microscopy. Changes in expression of slit pore constituents (podocin, CD2AP, nephrin and ZO-1), cytoskeleton-associated proteins (actin, -actinin, ezrin and synaptopodin), the GDH-podocyte adhesion molecules
3-integrin, and heparan sulfate were studied by immunofluorescence. FGS was scored by light microscopy at 3 weeks after induction of albuminuria.
Results. Albuminuria in Thy-1.1 transgenic mice was observed within 10 min after anti-Thy-1.1 mAb injection. This rapid development of albuminuria was accompanied by a reduction in number of podocytic foot processes from 20.0 ± 0.7/10 µm glomerular basement membrane (GBM) in saline-treated transgenic mice to 8.0 ± 0.5 and 2.2 ± 0.2 in anti-Thy-1.1-treated mice, at 10 min and 8 h after treatment, respectively. In addition, we observed a significant decrease in width of remaining slit pores, from 32.7 ± 1.1 to 26.8 ± 1.4 nm at 10 min after mAb injection. By immunofluorescence, we did not observe major changes in the expression pattern of any of the proteins studied. There was no correlation between the injected dose of the anti-Thy-1.1 mAb and the acute albuminuria. In contrast, the percentage of FGS at 3 weeks correlated with the dose, and a significant correlation between the percentage of FGS and the time-averaged albuminuria over the 3 week study period (P < 0.001) was found.
Conclusion. Injection of mAb directed against the Thy-1.1 protein, in young non-albuminuric Thy-1.1 transgenic mice, induced an acute albuminuria within 10 min, which was accompanied by foot process effacement. Notably, we observed a decrease in slit pore width although the expression of slit pore proteins was unchanged. Also, the acute albuminuria could not be related to alterations in cytoskeleton-associated proteins, the GBM adhesion molecule 3-integrin or heparan sulfate in the GBM. The dose-dependent development of FGS and the correlation between the percentage FGS and time-averaged albuminuria suggest that, in our model, FGS is a consequence of podocyte injury. However, the data leave open the possibility that albuminuria itself contributes to FGS development. The Thy-1.1 transgenic mouse model is an excellent model to study further the relationship between podocytic injury, albuminuria and the development of FGS.
Keywords: focal glomerulosclerosis; podocyte; proteinuria; Thy-1.1 transgenic mouse
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Introduction |
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Many models have been used to study proteinuria. We recently have reported detailed studies of acute proteinuria in a mouse model, the so-called Thy-1.1 transgenic mouse. The mousehuman chimeric transgene causes ectopic expression of the mouse Thy-1.1 antigen on podocytes. Injection of monoclonal antibody (mAb) directed against the Thy-1.1 antigen causes albuminuria within 24 h [5]. Also, induction of proteinuria, by injecting either anti-Thy-1.1 mAb or a combination of mAb directed against aminopeptidase A (APA), causes accelerated development of FGS in Thy-1.1 transgenic mice [5]. FGS is the characteristic lesion in progressive renal injury, and may be the consequence of longstanding proteinuria [6]. However, the relationship between proteinuria and FGS needs further study.
In the present study, we have evaluated the time course of proteinuria in the Thy-1.1 transgenic mouse. Next, we specifically have studied alterations in podocyte morphology, in slit pore geometry and expression of proteins involved in podocyteGBM adhesion that occur upon onset of proteinuria. In addition, we have questioned whether the acute proteinuria and the development of FGS are dose dependent and related.
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Subjects and methods |
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Antibodies
For in vivo experiments, a mouse anti-mouse Thy-1.1 mAb (19XE5: subclass IgG3) was used. 19XE5 was generated in vitro by hollow fibre culture, purified by protein A column affinity chromatography and concentrated (Nematology Department, Agriculture University Wageningen, The Netherlands). The mAb was decomplemented at 56°C for 45 min, sterilized by passage through a sterile 0.2 µm filter, and stored at -80°C. For immunohistological detection of glomerular localized antigens and injected antibodies, we have used the antibodies as listed in Table 1.
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Based on the obtained data (see Results), a dose of 1000 µg of anti-Thy-1.1 mAb in 0.9% saline solution (total volume 0.1 ml) was used in further experiments. To determine the time course of the acute proteinuria, urine samples were collected by bladder punctures at different time points (10 min, 1, 4, 8 and 24 h). After bladder puncture, mice were killed and kidneys were removed to be processed for histology. Transgenic mice injected with 0.9% saline solution alone were used as controls.
Urine albumin measurement
Urine albumin was measured by radial immunodiffusion using a goat antiserum against mouse albumin [8].
Normal physiological albuminuria was determined in wild-type (Thy-1.1 negative) mice (27 ± 2 µg/ml).
Light microscopy and immunofluorescence
For light microscopy, kidney fragments were fixed in Bouins solution, dehydrated, and embedded in paraplast (Amstelstad, Amsterdam, The Netherlands), and 4 µm sections were stained with periodic acidSchiff and methenamine silver [8]. To obtain the FGS score, at least 100 glomeruli were evaluated for the presence of podocytic hypertrophy, synechiae, sclerosis or hyalinosis, and the percentage of abnormal glomeruli was calculated.
For immunofluorescence (IF), kidney fragments were snap-frozen in liquid nitrogen, and 2 µm acetone-fixed cryostat sections were used. The expression of Thy-1.1 was studied by indirect IF by using the primary antibodies followed by fluorescein isothiocyanate (FITC)-labelled secondary antibodies as listed in Table 1. The expression of important constituents of the podocyte and the GBM was examined at different time points (10 min, 1 and 24 h) using unlabelled primary mAbs and FITC-labelled secondary antibodies as listed in Table 1. The slides were examined with a confocal microscope (Leica lasertechnik GmbH, Heidelberg, Germany).
For electron microscopy, small fragments of the kidney were fixed in 2.5% glutaraldehyde dissolved in 0.1 M sodium cacodylate buffer, pH 7.4 overnight at 4°C and washed in the same buffer. The tissue fragments were post-fixed in cacodylate-buffered 1% OsO4 for 2 h, dehydrated, and embedded in Epon812 (Merck, Darmstadt, Germany). Ultrathin sections were used and contrasted with 4% uranyl acetate for 45 min and subsequently with lead citrate for 4 min at room temperature. Sections were examined in a JEOL 1200 EX2 electron microscope (JEOL, Tokyo, Japan).
Immunoelectron microscopy
The localization of the Thy-1.1 antigen on podocytes was examined by indirect immunoelectron microscopy (IEM), using immunoperoxidase labelling on 20 µm frozen sections. A Thy-1.1 transgenic mouse was first perfused retrogradely via the aorta with phosphate-buffered saline (PBS) for 5 min and subsequently with a mixture of 10 mM periodate, 75 mM lysine and 2% paraformaldehyde, pH 6.2 (PLP) for 10 min. The kidneys were removed and small pieces were fixed further by immersion for 3 h in PLP. After rinsing several times in PBS, the fragments were cryoprotected by immersion in 2.3 M sucrose pH 7.2 for 1 h and then frozen in liquid nitrogen. Sections (20 µm thick) were rinsed in PBS for 1 h, then incubated with the anti-Thy-1.1 mAb diluted in PBS containing 1% bovine serum albumin (BSA) for 18 h at 4°C, followed, after several washes with PBS, by incubation with a peroxidase-labelled rabbit anti-mouse IgG (Dako Glostrup, Denmark) diluted in PBS containing 1% BSA. After three washes in PBS, the sections were incubated in PBS, pH 7.4 containing diaminobenzidine (DAB) medium for 10 min, followed by DAB with addition of 0.003% H2O2 for 7 min. The sections were washed in distilled water, post-fixed in palade buffer containing 1% OsO4 for 30 min at 4°C, dehydrated, and embedded in Epon812 (Merck, Darmstadt, Germany). Ultrathin sections were examined in a JEOL 1200 EX2 electron microscope (JEOL, Tokyo, Japan).
Measurement of foot processes, GBM and slit pores
Negatives of electron micrographs (magnification x6000) were scanned at 600 d.p.i. resolution using a scanner (Epson Perfection 1200 Photo, Epson Europe, Amsterdam), resulting in a specimen-level pixel size of 7 x 7 nm2. Measurement of the resulting images was performed using Leica QWin Pro V2.4 (Leica Imaging Systems Ltd, Cambridge, UK) under Microsoft Windows NT 4.0. The system was callibrated using the marker bar on the electron micrographs. For five open random capillary loops in each of five randomly selected glomeruli per specimen, the GBM was selected using a graphic tablet. The image analysis software was used to measure the length and width of the GBM of each loop. Also, for each loop, the number of podocytic foot processes was counted manually and expressed as the number of foot processes per 10 µm GBM length. Results of the measurement of 25 capillary loops were averaged. For each specimen, the width of
100 individual slit pores was measured using the same set of digitized electron micrographs. To obtain the slit pore width, the diameter of the narrowest region of the pore between two adjacent foot processes was measured.
Statistical analysis
Time-averaged albuminuria was calculated after log transformation. For multiple comparisons, ANOVA was used and post hoc analyses were done with Tukeys test. To test the homogeneity of variances, the Levene statistic was used. P < 0.05 was considered significant. For correlations, Pearsons r was calculated. All values are expressed as means ± SEM unless indicated otherwise.
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Results |
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Based on the above-mentioned experiments, we have used a dose of 1000 µg of anti-Thy-1.1 mAb for the following experiments. Results of albuminuria measured at 10 min, 1, 4, 8 and 24 h after anti-Thy-1.1 mAb administration are presented in Figure 2. Saline-injected transgenic mice showed no noteworthy albuminuria (93 ± 4 µg/ml). Injection of anti-Thy-1.1 mAb resulted in an acute albuminuria, which was observed already at 10 min after intravenous anti-Thy-1.1 mAb administration, and peaked after 4 h. Injection of anti-Thy-1.1 mAb into non-transgenic mice did not affect the normal physiological albuminuria (data not shown).
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Next we studied by IF changes in the expression pattern of slit pore constituents (podocin, CD2AP, nephrin and ZO-1), cytoskeleton-associated proteins (actin, -actinin, ezrin and synaptopodin), GBMpodocyte adhesion molecule (
3-integrin) and heparan sulfate. For most of these proteins, we did not observe any changes in expression pattern upon anti-Thy-1.1 mAb injection. However, the homogeneous binding pattern of antibodies to podocin changed into a fine granular binding at 1 h after anti-Thy-1.1 mAb injection. At 4 h after mAb injection, binding was more granular and there was a slight reduction in signal intensity (Figure 6). We did not observe any difference in the expression of the slit pore constituents, cytoskeleton-associated proteins or adhesion molecules at the early time point of 10 min after anti-Thy-1.1 mAb injection.
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Discussion |
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Therefore, in addition to podocyte morphology, we have in the present study focused on the expression of proteins involved in maintaining the cytoskeleton of the podocyte, in constituents of the slit pore and in molecules involved in podocyteGBM adhesion.
We have performed detailed electron microscopic measurements of podocytic foot processes and slit pore width. We did not observe large gaps between podocytes nor podocyte detachment. In this respect, our mouse model closely resembles human nephropathies such as minimal change disease or FGS. Our model contrasts with two other widely used models of podocyte injury, proteinuria and progressive glomerulosclerosis using the chemotherapeutic agents, puromycin aminonucleoside and adriamycin. These agents cause severe podocytic injury leading to necrosis and detachment of the podocytes from the GBM. In both models the level of proteinuria correlates best with the degree of podocyte detachment [11]. Thus, in our model, the podocyte injury is relatively small, with no detachment detectable in the first 24 h after mAb injection, despite massive proteinuria.
We noted that already at 10 min after mAb injection, albuminuria was accompanied by a major decrease in the number of foot processes. We feel that this finding suggests that foot process effacement is a consequence of podocyte injury rather than a mere consequence of the albuminuria. Admittedly, because of the abrupt onset of albuminuria in this model, we cannot discern the precise relationship in time between foot process effacement and albuminuria. We previously have studied the relationship between foot process effacement and albuminuria in a different mouse model. In the latter model, albuminuria is induced by a single injection of a combination of two mAbs directed against APA, a protein expressed in the cell membrane of podocytes and of the brush border of proximal tubuli [12]. After administration of the anti-APA mAbs, the first sign of proteinuria was detected at 6 h, whereas the morphometric measurements showed a reduction in the number of foot processes already after 4 h [13]. These findings suggest that foot process effacement is a marker of podocyte injury or activation and not a consequence of albuminuria. This latter conclusion is supported by the observation that reactive oxygen metabolites can cause massive proteinuria, which is not accompanied by ultrastructural abnormalities [14]. A causeeffect relationship between foot process effacement and albuminuria remains to be proved. In fact, in the APA model, maximal effacement was measured at 24 h after anti-APA mAb injection while, at this time point, albuminuria had almost returned to baseline level. Thus, both foot process effacement and albuminuria may be consequences of podocyte injury and are not necessarily causally related.
We noted a significant reduction in slit pore width already at 10 min after anti-Thy-1.1 mAb injection. The reduction of slit pore width may be explained by the collapse of the glomerular tuft after injection of the mAb. However, ischaemic changes of the glomeruli were not observed at the earliest time points. Alternatively, this reduction in slit pore diameter may point to the presence of intrinsic injury with alterations in the interactions of slit diaphragm proteins such as podocin, nephrin and CD2AP. However, we were unable to demonstrate major changes in the expression pattern of these slit diaphragm proteins as observed in IF studies. Admittedly, these studies cannot exclude that structural changes in these proteins might have occurred.
We have studied several GBM characteristics in more detail. We did not observe reduced expression of 3-integrin, a molecule involved in podocyteGBM adhesion. This finding seems in accordance with the fact that we never observed podocyte detachment in electron microscopy. There was also no evidence of loss of GBM negative charge, an important mechanism of proteinuria in other models, ascribed to destruction of glycosaminoglycans by reactive oxygen species. In our model, the expression of JM403 and HS4C3, antibodies against heparan sulfate which are used to characterize GBM negative charge [15,16], remained normal, which indicates that the acute albuminuria is not related to a defect or reduction of GBM negative charge. We did not test the possible role of vasodilatory and permeability-promoting factors such as vascular endothelial growth factor (VEGF), platelet-activating factor (PAF) and tumour necrosis factor-
(TNF-
), which can be produced by podocytes [17,18], Thus, we cannot exclude these factors as mediators of proteinuria.
In our model, the development of FGS was dose dependent. This finding fits with the recently proposed role of podocyte injury in FGS. FGS is considered to be the consequence of loss of podocytes due to podocyte injury [19]. Since podocytes are terminally differentiated cells and unable to replicate, lost podocytes cannot be replaced. The naked GBM will be covered by parietal cells and Bowmans membrane, giving rise to adhesions, the first step in the formation of a sclerotic lesion. In our model, higher doses of antibodies will lead to more extensive podocyte injury, thus explaining the dose dependency of the FGS. One could argue that there was no clear relationship between the antibody dose and the level of the acute albuminuria. However, our data indicate that the acute albuminuria might not be the best reflection of podocyte injury. Even with a relatively low dose, massive acute albuminuria occurred, which plateaued, most probably as a consequence of altered glomerular haemodynamics as indicated by the development of oliguria. We observed a strong correlation between time-averaged albuminuria and the number of FGS lesions. Most probably the persistence of albuminuria is a better reflection of the severity of podocyte injury than the acute albuminuria. Although we feel that in our model FGS is linked to podocyte injury, the data leave open the possibility that albuminuria itself is detrimental to podocytes and contributes to FGS development. This, however, needs to be investigated further.
The mechanisms that induce the podocyte alterations upon mAb injection are still unknown. Thy-1.1 is a 17 kDa cell surface glycoprotein, anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor [20]. Recent studies provided evidence that GPI-anchored proteins including Thy-1.1 are partitioned in lipid rafts [21]. These lipid rafts are proposed to be involved in membrane trafficking of GPI-anchored proteins and in signal transduction via src-family kinases [22]. Lipid raft components can be cross-linked with antibodies or lectins in living cells, resulting in coalescence of individual rafts to form raft clusters. Several studies showed that antibody-mediated cross-linking of Thy-1 and other GPI-anchored proteins can induce cell activation through activation of src-family kinases [23]. Antibody-mediated cross-linking of gangliosides, which together with cholesterol form lipid rafts, can mimic GPI-anchored protein signalling, illustrating the importance of the raft structure [24]. Interestingly, Reivinen et al. [25] described a podocytic-specific ganglioside in rat kidney, O-acetylated GD3. In vivo binding of antibodies directed against this ganglioside induced rapid morphological changes in the foot processes [26]. Recent studies showed that lipid raft domains are also involved in the spatial organization and the signalling events of the glomerular slit diaphragm [27]. Furthermore, Fyn, a member of the src-kinase family, binds to and phosphorylates nephrin [28]. This phosphorylation might be a part of the nephrin-dependent signalling, which is believed to be essential for podocyte integrity [28,29]. It is tempting to speculate that co-localization of the Thy-1.1 protein and slit diaphragm proteins in raft domains plays a role in the podocyte alterations observed in the anti-Thy-1.1 mAb-injected Thy-1.1 transgenic mice.
Besides activating a specific signalling cascade, the podocytic alterations in the Thy-1.1 mouse can also be caused by non-specific injury due to antibody binding. Clustering of rafts carrying the Thy-1.1 protein might influence, for instance, the location and behaviour of other surface proteins, influencing podocyte and slit diaphragm geometry, without protein signalling. Furthermore, the net negative charge is thought to be necessary for maintaining the integrity of the slit pores [30]. Lowering the high negative charge by injecting protamine sulfate induces a rapid (15 min) effacement of the foot processes [31]. Isoelectric focusing revealed that the anti-Thy-1.1 mAb has a pI of 8. This pI results, in a physiological environment, in a net positive charge. Binding of a large amount of this antibody can neutralize the podocytic negative charge, resulting in the foot process effacement.
In conclusion, a single injection of the anti-Thy-1.1 mAb in Thy-1.1 transgenic mice induces an acute and massive albuminuria, which is followed by an accelerated FGS. The early albuminuria is already accompanied by foot process effacement and narrowing of the slit pore. Although we did not observe changes in the expression of important podocyte- and cytoskeleton-associated molecules, it is possible that structural changes in such molecules are involved. In the Thy-1.1 model, FGS lesions developed within 3 weeks in a dose-dependent process. The immediate onset of the albuminuria together with the fast development of FGS makes the Thy-1.1 model an ideal model for further studies of the relationship between podocytic injury, albuminuria and FGS development.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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