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From the Dipartimento di Medicina Interna,*
Università di Torino, Torino, Italy; the Dipartimento di Medicina
Interna,
Cattedra di Nefrologia,
Università di Genova, Genova, Italy; the Dipartimento di Scienze
Cliniche e Biologiche,
Università
dell’Insubria, Varese, Italy; the Department of
Biochemistry,
University of Oulu, Oulu,
Finland; and the Department of Medical Biochemistry and
Biophysics,¶
Karolinska Institute,
Stockholm, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and puromycin, induced the shedding of
nephrin with a loss of surface expression. This phenomenon was
abrogated by cytochalasin and sodium azide. These results suggest that
the activation of cell cytoskeleton may modify surface expression of
nephrin allowing a dislocation from plasma membrane to an extracellular
site.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The aim of the present study was to investigate whether the expression of nephrin was altered in biopsies from patients with primary acquired nephrotic syndrome. Because we found that in these patients the immunohistochemical-staining pattern of nephrin was severely altered, we also studied the effect of various stimuli on the surface expression of nephrin on human cultured podocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mouse anti-nephrin antibody (IgG1) is a
mAb specific for the extracellular fibronectin type III-like motif of
the recombinant human nephrin produced in A293 cells ( V. Ruotsalainen
and K. Tryggvason, manuscript in preparation). In Western blots,
nephrin antibody recognized the extracellular domain of recombinant
human nephrin and a 180-kd protein in lysates of human
glomeruli.3
An irrelevant IgG1
isotypic control antibody was purchased from Cedarlane (Hornby, Ontario
Canada). Rabbit antiserum to human-IgG was purchased from Dade Behring
(Marburg, Germany). Fluorescein isothiocyanate (FITC)-conjugated sheep
anti-mouse IgG (adsorbed with human serum proteins);
tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat
anti-rabbit IgG (adsorbed with human IgG); FITC-phalloidin; human
IgG4 kappa (IgG4); human
tumor necrosis factor- (TNF-); cytochalasin B; sodium azide; C5,
C6, C7, C8, and C9 complement fragments; methionine; chloramine-T; and
puromycin were obtained from Sigma Chemical Co. (Saint Louis, MO).
Mouse mAb anti-human C9 neoantigen of the C5b-9 complex (MAC) was
purchased from DAKO (Glostrup, Denmark). The antibody anti-E-selectin
was obtained from Chemicon Int. (Temecula, CA). Vectashield
mounting medium was purchased from Vector Laboratories, Inc.
(Burlingame, CA).
Patients
The study included 30 proteinuric patients (27 of 30 with
nephrotic syndrome) and six patients with minimal proteinuria.
Nephrotic syndrome was defined as proteinuria 
3.5 g/day and serum
albumin 
3.0 g/dL. The histological diagnosis of proteinuric patients
was membranous GN in 13 cases, minimal change GN in 10 cases, and focal
segmental glomerulosclerosis (FSGS) in seven cases. Patients with
minimal proteinuria selected for the study presented IgA GN. None of
the patients had evidence of systemic disease on a clinical or
laboratory basis. Table 1
depicts some of
the clinical features of the patients included in the study. As
control, 10 specimens were obtained from normal kidney portions of
patients undergoing surgery for cancer. Patients were selected for
absence of proteinuria and lack of glomerular abnormalities detected by
light and immunofluorescence microscopy.
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Culture of Glomerular Epithelial Cells
Decapsulated glomeruli were isolated by differential sieving from renal cortex fragments taken from surgically removed kidneys of five Caucasian patients.9,10 Primary cultures of glomerular epithelial cells (GECs) were established as previously described.11 GECs were obtained by plating at high density glomeruli untreated with collagenase. After a 10-day incubation in Dulbecco’s modified Eagle’s medium containing 20% fetal calf serum, the cultures were trypsinized and the outgrowing GECs were expanded. Phenotypic characterization was performed according to cell morphology (polyhedral cells with cobblestone-like appearance); positive staining for synaptopodin, podocalyxin, zonula occludens-1 (ZO-1), cytokeratin, vimentin, and laminin; negative staining for smooth muscle-type myosin, FVIIIr:Ag, and CD45; cytotoxicity in response to puromycin aminonucleoside (10 to 50 µg/ml).10,12-14
Established lines of differentiated GECs were obtained by infection of pure primary cultures with a hybrid Adeno5/SV40 virus as previously described.11,15 Individual foci of immortalized cells were subcultured and cloned. The selected clones were used between passages 25 and 40. The GEC line used in the present study was characterized as previously described according to the phenotype, following the criteria mentioned above.11,13
Immunofluorescence Studies
Immunofluorescence (IF) studies were performed on kidney biopsies from the patients described above. The tissues were rapidly frozen in liquid nitrogen, and 2-µm-thick cryostat sections were fixed in 3.5% paraformaldehyde for 15 minutes and washed in phosphate-buffered saline (PBS). The sections were incubated with anti-nephrin mAb at a concentration of 10 µg/ml or with the irrelevant mouse IgG1 isotypic control antibody, for 2 hours at room temperature, washed in PBS, and incubated with FITC-conjugated sheep anti-mouse IgG. Double staining was performed on biopsies from patients with membranous GN using a rabbit antiserum to human IgG and a TRITC-conjugated goat anti-rabbit IgG. A rabbit nonimmune serum was used as irrelevant control.
IF on cultured GECs was performed as previously described.9 Briefly, coverslip-attached GECs at subconfluent density were fixed in 3.5% paraformaldehyde containing 2% sucrose for 15 minutes at room temperature and washed in PBS. In experiments aimed to study intracellular localization of nephrin or cell cytoskeleton, cells were made permeable to large molecules by soaking coverslips for 5 minutes at 0°C in HEPES-Triton X-100 buffer (20 mmol/L HEPES, pH 7.4, 300 mmol/L sucrose, 50 mmol/L NaCl, 3 mmol/L MgCl2, and 0.5 Triton X-100). Cells were then incubated with antibodies as described for the biopsies. The slides were then washed, mounted with Vectashield mounting medium, and examined.
Control experiments included incubation of sections or cells with nonimmune isotypic control antibodies or the omission of primary antibodies followed by the appropriate labeled secondary antibodies. The specificity of anti-nephrin mAb was tested by pre-adsorption of the antibody (10 µg/ml) with purified recombinant extracellular nephrin (30 µg/ml).
The number of glomeruli available on each biopsy for analysis of nephrin expression ranged between 3 to 7. Three nonsequential sections were examined for each specimen. Nephrin expression was analyzed semiquantitatively by measuring fluorescence intensity by digital image analysis (Windows MicroImage, version 3.4 CASTI Imaging, Venezia, Italy) of images obtained using a low-light video camera (Leica DC100, Wetzlar, Germany) on a 180-µm diameter field. The results were expressed as relative fluorescence intensity on a scale from 0 (fluorescence of background of tissue) to 255 (fluorescence of standard filter).
Detection of Nephrin mRNA Expression by Reverse Transcriptase (RT)-Polymerase Chain Reaction (PCR)
RT-PCR was performed using total RNA from GECs. Total RNA was extracted using Tri Reagent (Sigma) and precipitated with isopropanol. Complementary DNA was obtained by using oligo-p(dT)15 primers (Boehringer Mannheim, Mannheim, Germany). Reverse transcription was performed at 42°C for 60 minutes; in addition to 1 µg of RNA, the reaction mixture (20 µl) contained 10 mmol/L Tris, 50 mmol/L KCl, pH 8.3, 5 mmol/L MgCl2, 1 mmol/L dNTPs, 50 U RNase inhibitor and 20 U AMV reverse transcriptase (Boehringer Mannheim). For reverse transcriptase-negative controls, the enzyme was omitted. cDNA was then subjected to 35 cycles of amplification by the PCR in an automated DNA thermal cycler (Hybaid, Ashford, UK). For detection of human nephrin and human glyceraldehyde phosphate dehydrogenase (h-GAPDH), used as housekeeping gene, sequence-specific oligonucleotide primers (purchased from TIB Molbiol, Genova, Italy) were designed (nephrin: 3'reverse, TAC ACC AGA TGT CCC CTC AG; 5' forward, TCT TAT TCC CGA GGT TTC AC; GAPDH: 3'reverse, TCT AGA CGG CAG GTC AGG TCC ACC; 5' forward, CCA CCC ATG GCA AAT TCC ATG GCA). The PCR mixture (50 µl) contained 10 mmol/L Tris-HCl, pH 8.8, at 25°C, 50 mmol/L KCl, 0.1% Triton X-100, 1.25 mmol/L MgCl2, 0.2 mmol/L dNTPs, 25 pmol of (+) and (-) primers, and 1.25 U thermostable DNA polymerase (Finnzymes). Times and temperatures for denaturation, annealing, and extension were 1 minute at 95°C, 1 minute at 60°C, and 3 minutes at 72°C, respectively. Amplification products (257 bp) for nephrin and (598 bp) for GAPDH were visualized by ethidium bromide staining after agarose gel electrophoresis.
Detection of Nephrin Expression by Western Blot Analysis
Protein concentration of GEC lysates obtained as previously described16 was determined by the Bradford technique, and the protein content of the samples was normalized to 100 32 µg/sample by appropriate dilution with Laemmli buffer. Proteins were directly subjected to 8% SDS-polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose. The filters were incubated with blocking solution (10% low-fat milk in 20 mmol/L Tris/HCl, pH 7.6, and 17 mmol/L NaCl) for 60 minutes. The anti-nephrin mAb was then added at a concentration of 2.5 µg/ml, and the incubation was performed overnight at 4°C. For detection, the filters were washed four times (15 minutes each wash) with PBS and 0.5% Tween 20, and reacted for 60 minutes at room temperature with peroxidase-conjugated protein A (200 ng/ml; Amersham, Buckinghamshire, UK). The enzyme was removed by washing as above. The filters were incubated for 2 minutes with a chemiluminescence reagent (ECL, Amersham) and exposed to an autoradiography film for 1 to 5 minutes.
As negative control for the expression of nephrin, an immortalized cell line of human renal tubular epithelial cells was used.15
Experimental Design
The expression of nephrin by GECs was evaluated by indirect IF on cells fixed with paraformaldehyde followed or not by permeabilization with Triton X-100. In some experiments, living (unfixed) GECs were incubated at 37°C or 4°C for 60 minutes with anti-nephrin mAb to evaluate antibody-induced antigen redistribution.9 To study in vitro the role of immune complexes on the surface expression and redistribution of nephrin, GECs were incubated with human aggregated IgG4 (agIgG4) (1 µg/ml) in Dulbecco’s modified Eagle’s medium and 10% fetal bovine serum for 60 minutes at 37°C before fixation and staining with anti-nephrin mAb and rabbit anti-human-IgG. AgIgG4 was obtained by heating at 63°C for 30 minutes, as described.17 As control, cells were incubated with human disaggregated IgG4 in Dulbecco’s modified Eagle’s medium and 10% fetal bovine serum for 60 minutes at 37°C before staining with antibodies. Human IgG4 were disaggregated after centrifugation for 4 hours at 100,000 x g at 4°C.18
Actin microfilament alterations in
agIgG4-stimulated GECs were evaluated by
FITC-phalloidin staining after permeabilization of the cells. Other
stimuli, such as TNF-, puromycin, or MAC, known to be involved in
the pathogenesis of GN and to affect the cell cytoskeleton, were used
to evaluate the role of actin microfilament alterations on the
expression and redistribution of nephrin. Cells were incubated with
human TNF- (10 ng/ml) or puromycin (5 µg/ml) in Dulbecco’s
modified Eagle’s medium and 10% fetal bovine serum for 1 hour at
37°C before staining with anti-nephrin mAb. Actin microfilament
alterations in TNF- or puromycin-stimulated GECs were evaluated as
FITC-phalloidin staining. Assembly of the MAC on GECs was performed as
previously described.19
Briefly, nonenzymatic formation of
a C5b-like C5C6 complex was obtained by incubation of 10 µg of C5 in
10 µl of veronal buffer with 10 µl of 0.32 mmol/L chloramine-T for
10 minutes at room temperature. After inactivation of chloramine-T with
10 µl methionine (1 mmol/L), purified C6 (20 µg) was added in 300
µl of serum-free medium and incubated for 24 hours at 37°C. The
assembly of the MAC into cell plasma membrane was obtained by 15
minutes of pre-incubation (37°C) of C7 (10 µg/ml) with the C5b-C6
complex (5 µg/ml). After two washings with serum-free medium, C8 (10
µg/ml) and C9 (10 µg/ml) complement components were added and
incubated for 30 minutes at 37°C. In selected experiments heat
inactivated (100°C for 30 minutes) or polymyxin B-treated (5 to 50
µg/ml) complement components were used as control. After cell
fixation, the expression of nephrin was evaluated by IF with
anti-nephrin mAb. In parallel experiments the insertion of MAC into GEC
plasma membrane was assessed by IF using a mAb anti-human C5b-9
reacting with a neoepitope exposed only in activated C9 in the
solid-phase membrane form and in the fluid form of MAC, but not in
native C9.20
In some experiments, before incubation with the various stimuli, the GECs were pre-incubated for 15 minutes with drugs that interfere with cytoskeletal function such as cytochalasin B (5 and 10 µg/ml) or with cell metabolism such as sodium azide (10-3 to 10-1 mol/L). The cell viability was assessed by trypan blue exclusion.
Statistical Analysis
All results are given as mean values ± SD. Differences between multiple groups were analyzed by one-way analysis of variance in combination with Tukey’s multiple comparison test. Linear regression analysis was performed between relative fluorescence intensity for nephrin and extent of proteinuria. A P value of <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of nephrin was evaluated by indirect IF in 30
proteinuric patients (27 with nephrotic syndrome) including 13
membranous GN, 10 minimal change GN, and seven FSGS, and in six
patients with IgA GN and minimal proteinuria (Table 1)
using mAb
specific for the extracellular domain of nephrin. As control, 10
specimens of normal kidney portions obtained from patients undergoing
surgery for renal tumors were used.
In normal controls, nephrin exhibited a glomerular epithelial pattern
with a punctate/linear distribution along the peripheral capillary
loops (Figure 1A)
. In glomeruli of
patients with membranous GN, a more granular pattern or a loss of
staining of nephrin was observed (Figure 1B)
. Figure 1, C and D
, shows
the co-localization of nephrin and IgG evaluated by double IF in the
same glomerulus of a patient with membranous GN. In Figure 1C
, the
green fluorescence depicts the distribution of nephrin. The red
fluorescence observed in Figure 1D
shows the granular distribution of
IgG along the glomerular basement membrane. The overlap of staining for
nephrin (green) and IgG (red) results in a yellow staining of granular
deposits in the merge (Figure 1D)
. A granular pattern with aspects of
apparent plasmalemmal dislocation from the normal expression sites of
nephrin and a loss of staining were also observed in patients with
minimal change GN (Figure 2, A and B)
and
FSGS (Figure 2C)
. In IgA GN, the staining pattern of nephrin showed an
epithelial distribution, similar to that of controls (Figure 2D)
. As
shown in Figure 3
, relative fluorescence
intensity was significantly reduced in all proteinuric patients
irrespective of the primary disease. In contrast, the fluorescence
intensity in glomeruli of patients with IgA GN with minimal proteinuria
did not significantly differ from that of controls (Figure 3)
. The
average variability in nephrin staining pattern from one glomerulus to
the next within individual patients and controls was 20.6 ± 16
and 14.4 ± 4, respectively.
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an inverse
correlation between relative fluorescence intensity and extent of
proteinuria was detected when all patients with GN were analyzed. In
contrast, when linear regression analysis was performed only on
patients with nephrotic syndrome, no significant correlation was
observed.
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Expression of Nephrin by GECs
Primary and immortalized GECs showed expression of the nephrin
mRNA and of the protein by RT-PCR (Figure 5A)
and Western blot analysis (Figure 5B)
, respectively. When tested by indirect IF, antibody specific for
the extracellular domain of nephrin bound to GECs unfixed or fixed with
paraformaldehyde with a punctate granular pattern, suggesting a surface
expression of nephrin (Figure 6A)
. In
paraformaldehyde-fixed and permeabilized GECs a combination of fine
granular and diffuse staining was observed suggesting the presence of
an intracellular pool of nephrin. In permeabilized cells the staining
was more evident around the nuclei (data not shown).
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Experiments of nephrin redistribution were performed on
immortalized GECs. When living (unfixed) GECs were incubated with
anti-nephrin mAb for 60 minutes at 37°C, but not at 4°C, the
binding was rapidly converted from fine granular into a patchy pattern,
suggesting antibody-induced antigen redistribution (data not shown).
Therefore, we tested whether agIgG4 were able to
change the surface expression of nephrin. Incubation of living GECs at
4°C for 60 minutes with agIgG4 resulted in
diffuse surface binding of IgG4 to GECs (data not
shown). When incubated at 37°C for 60 minutes,
agIgG4 appeared redistributed in one or multiple
aggregates (capping) (data not shown). When cells were fixed with
paraformaldehyde after 60 minutes incubation with
agIgG4, nephrin appeared focally redistributed on
the cell surface, leaving variable parts of the cell surface devoid of
antigen (Figure 6B)
. Double staining of nephrin and IgG showed focal
co-localization on the cell surface (Figure 6B
, inset). In most GECs
the disappearance of nephrin from the surface was predominant, as
indicated by a significant decrease of IF staining (Figures 6C and 7A)
,
suggesting shedding of the protein from the cell surface.
AgIgG4 induced disruption of normal cellular
organization of F-actin (Figure 6D)
in GECs, with loss of stress fibers
and peripheral actin localization (Figure 6, E and F)
. In contrast,
disaggregated IgG4 did not induce nephrin
redistribution and disappearance (Figure 6G)
or cytoskeletal changes
(data not shown). We therefore investigated whether the observed
nephrin redistribution could be ascribed to cytoskeletal reorganization
induced by agIgG4. Plasma membrane insertion of
MAC, TNF-, or puromycin, stimuli known to affect the cytoskeleton of
GECs,14,21,22
also induced a significant reduction of IF
intensity (Figure 7A)
and a staining
pattern compatible with surface patching and shedding of nephrin
(Figure 6, H and I)
. Within 24 hours of removal of TNF- from the
culture medium, nephrin was fully re-expressed on GECs, indicating that
the effect of this cytokine was not to be ascribed to a cytotoxic
effect (Figure 6J)
. Moreover, the process of nephrin redistribution was
an active energy-requiring process, because sodium azide, an inhibitor
of oxidative phosphorylation and of glycolysis, prevented patching and
shedding induced by agIgG4, TNF-, and
puromycin (Figures 6K and 7B)
. Similar results were obtained with
cytochalasin B, a compound that affects the microfilaments of the
microtubular system,23
which prevented reduction of
fluorescence intensity and morphological evidences of nephrin
redistribution (Figures 6L and 7C)
.
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In control experiments, the staining for nephrin was completely abrogated by pre-adsorption of the antibody with the purified human recombinant extracellular nephrin. When the relevant antibodies were substituted with the nonimmune isotypic control antibodies or with the appropriate labeled secondary antibodies without the primary antibody the immunofluorescence was always negative (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Evidences for a critical role of nephrin in maintaining glomerular permeability in acquired proteinuric diseases was first derived from experiments in rats injected with mAb 5-1-6 that has been shown to be directed at the extracellular domains of nephrin.6 This treatment induced proteinuria and nephrotic syndrome.6 Moreover, a decrease in nephrin mRNA expression and redistribution of the protein were observed in several animal models such as puromycin aminonucleoside nephrosis and mercuric chloride GN.7,8
The present results demonstrated that, in patients with acquired nephrotic syndrome, the IF staining for nephrin was significantly reduced in intensity with extensive loss and granular redistribution. In membranous GN, granular deposits of nephrin were co-localized with the extracellular immune deposits. Moreover, granular distribution of nephrin suggesting a plasmalemmal dislocation from the normal expression sites at the interpodocytes filtration slits was also observed in minimal change GN and FSGS. Similar results have been reported in experimental animal models such as puromycin aminonucleoside nephrosis,7 mercuric chloride-treated rats,8 and nephritis induced by injection of mAb 5-1-6,6 in which the pattern of nephrin IF staining shifted from epithelial/linear to granular. In the present study we did not observe significant differences in the reduction of glomerular staining for nephrin among membranous GN, minimal change GN, and FSGS. In patients with IgA GN with minimal proteinuria neither a significant loss of IF staining nor a redistribution of nephrin were observed. This suggests that the reduced expression of nephrin was not related to a specific glomerular disease but rather to the proteinuric state. A decrease in glomerular expression of nephrin mRNA has been recently reported in one case of membranous GN and three cases of minimal change GN.26 These results suggested that nephrin may be a target of injury in acquired proteinuric diseases. However, it remains to be determined whether this was the cause of proteinuria or the consequence of podocyte injury.
In the present study we investigated whether different stimuli may
influence surface distribution of nephrin on glomerular podocytes. The
interaction of nephrin with specific antibodies induced a
redistribution of immune complexes formed on the cell surface with
patching and shedding of immune complexes containing nephrin. This led
to temporary disappearance of the antigen from the cell surface. This
process is reminiscent of antibody-induced redistribution of Heymann
antigen of the surface of cultured podocytes.9,27
We
therefore investigated whether addition to GECs of preformed immune
complexes mimicked by agIgG4 induced the
modulation of surface expression of nephrin. IgG4
was chosen because it is the most represented immunoglobulin subclass
in membranous GN.28
The results obtained indicate that
agIgG4 induced focal redistribution and extensive
loss of nephrin on the cell surface in association with changes in the
cytoskeleton organization. Because disaggregated
IgG4 did not stimulate nephrin redistribution,
one can speculate that the altered distribution of nephrin is not
because of a heterotopic association between nephrin and
IgG4, but rather to the interaction of
agIgG4 with the specific neonatal Fc receptor,
expressed by GECs.29
Fc receptor stimulation may induce
cytoskeletal rearrangement and consequent redistribution of nephrin
that seemed to be connected with actin.30
Indeed, other
stimuli affecting cytoskeleton organization, such as MAC, TNF-, and
puromycin, induced redistribution and loss of nephrin from the cell
surface. The role of the cytoskeleton was also confirmed by the effect
exerted by cytochalasin B, which disorganizes
microfilaments,23
preventing nephrin redistribution on the
surface of podocytes. This result, together with the recent report on
the role of nephrin in podocyte morphology,30
suggests
that stimuli affecting cytoskeleton organization may induce also
redistribution and shedding of nephrin from the surface of podocytes.
The inhibitory effect of sodium azide indicates that this process is
energy-dependent. Sodium azide inhibits oxidative phosphorylation and
glycolysis, and prevents capping and shedding in both
lymphocytes31
and podocytes.9
In conclusion, although the observations made with cultured GECs must be interpreted cautiously because of the obvious difference between the organization of podocytes in glomeruli and in culture, one can speculate that the reduction of nephrin protein expression observed in glomeruli of patients with acquired primary nephrotic syndrome may be the consequence of a podocyte injury triggered by different stimuli, such as antibodies, immune complexes, terminal components of complement, and cytokines.
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Footnotes |
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Supported by the National Research Council, Targeted Project Biotechnology and by Ministero Università e Ricerca Scientifica e Tecnologica (MURST) ex60%. Dr. Sophie Doublier is recipient of a Marie Curie individual fellowship from the European Community.
Accepted for publication February 5, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 Y. Xing, J. Ding, Q. Fan, and N. Guan Diversities of Podocyte Molecular Changes Induced by Different Antiproteinuria Drugs. Experimental Biology and Medicine, May 1, 2006; 231(5): 585 - 593. [Abstract] [Full Text] [PDF]
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C. Herve and J. Dantal Possible new perspectives for our understanding of nephrotic syndrome recurrence Nephrol. Dial. Transplant., January 1, 2006; 21(1): 10 - 13. [Full Text] [PDF]
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A. V. Cybulsky, R. J. Quigg, and D. J. Salant Experimental membranous nephropathy redux Am J Physiol Renal Physiol, October 1, 2005; 289(4): F660 - F671. [Abstract] [Full Text] [PDF]
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F. Nakhoul, R. Ramadan, E. Khankin, A. Yaccob, Z. Kositch, M. Lewin, S. Assady, and Z. Abassi Glomerular abundance of nephrin and podocin in experimental nephrotic syndrome: different effects of antiproteinuric therapies Am J Physiol Renal Physiol, October 1, 2005; 289(4): F880 - F890. [Abstract] [Full Text] [PDF]
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B. Bussolati, M. C. Deregibus, V. Fonsato, S. Doublier, T. Spatola, S. Procida, F. Di Carlo, and G. Camussi Statins Prevent Oxidized LDL-Induced Injury of Glomerular Podocytes by Activating the Phosphatidylinositol 3-Kinase/AKT-Signaling Pathway J. Am. Soc. Nephrol., July 1, 2005; 16(7): 1936 - 1947. [Abstract] [Full Text] [PDF]
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P. Gerke, L. Sellin, O. Kretz, D. Petraschka, H. Zentgraf, T. Benzing, and G. Walz NEPH2 Is Located at the Glomerular Slit Diaphragm, Interacts with Nephrin and Is Cleaved from Podocytes by Metalloproteinases J. Am. Soc. Nephrol., June 1, 2005; 16(6): 1693 - 1702. [Abstract] [Full Text] [PDF]
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M. Nangaku, S. J. Shankland, and W. G. Couser Cellular Response to Injury in Membranous Nephropathy J. Am. Soc. Nephrol., May 1, 2005; 16(5): 1195 - 1204. [Abstract] [Full Text] [PDF]
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