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Mario Negri Institute Bergamo, Italy
Ospedali Riuniti di Bergamo, Azienda Ospedaliera, Bergamo,
Italy
Beth Israel Deaconess Medical Center and Harvard Medical School, Boston,
Massachusetts.
Correspondence to Dr. Ariela Benigni, Mario Negri Institute for Pharmacological Research, Via Gavazzeni 11, 24125 Bergamo, Italy. Phone: 39-035-319-888; Fax: 39-035-319-331; E-mail: abenigni{at}marionegri.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At the end of the study, systolic BP, urinary protein excretion, and serum creatinine were measured. Rats were then anesthetized and kidneys were removed for histology, in situ hybridization, and immunohistochemistry for nephrin. In PHN animals at day 7 and month 8, immunohistochemistry was performed for the tight junction protein ZO-1, another component of the slit diaphragm, which seems to play a functional role in maintaining functional properties of the epithelial slit diaphragms (7).
Systolic BP was recorded by tail plethysmography in conscious rats (8). Twenty-four-h samples were collected using metabolic cages, and proteinuria was determined by modified Coomassie blue G dye-binding assay for proteins with bovine serum albumin as standard (9). Blood was collected from the tail vein of anesthetized animals. Serum was obtained after whole-blood clotting and kept frozen at -20°C until assayed. Creatinine was measured by the alkaline picrate method (10).
Histology
The removed kidneys were fixed for 6 h in Dubosq-Brazil, dehydrated in
alcohol, and embedded in paraffin. Kidney samples were sectioned at 3-µm
intervals, and three sections per slide were mounted. Sections were stained
with Masson's trichrome, hematoxylin and eosin, and periodic-acid Schiff
reagent (PAS stain). Sections including superficial and juxtamedullary
glomeruli were evaluated. Tubular (atrophy, casts, and dilation) and
interstitial changes (fibrosis and inflammation) were graded from 0 to 4+ (0,
no changes; 1+, changes affecting less than 25% of the sample; 2+, changes
affecting 25 to 50% of the sample; 3+, changes affecting 50 to 75% of the
sample; 4+, changes affecting 75 to 100% of the sample). At least 100
glomeruli were examined for each animal, and the extent of glomerular damage
was expressed as the percentage of glomeruli presenting focal or global
sclerotic lesions. All renal biopsies were analyzed by the same pathologist,
who was unaware of the nature of the experimental groups.
Cloning of Mouse cDNA
Total cellular RNA was purified from 6-wk-old C57B1/6J mouse kidneys using
TRIzol Reagent (Life Technologies, Gaithersburg, MD) according to the
manufacturer's directions. One µg of RNA was reverse-transcribed with
Superscript II reverse transcriptase (Life Technologies) and oligo-dT primer.
The resulting cDNA was treated with RNase H and subjected to 35 cycles of PCR
with the primers created based on exon sequence derived from genomic DNA
sequence. Primers used were 5'-ATG AGA CAG TCA TGG ATG GC-3' and
5'-TCT CTC CAC CTC GTC ATA CAG-3', corresponding to nucleotides
1154 to 1173 and 3526 to 3546, respectively. PCR reaction was performed as
follows: denaturation at 94°C for 30 s, annealing at 58°C for 30 s,
and extension at 72°C for 3 min using Expand High Fidelity enzyme cocktail
(Roche Molecular Biochemicals, Indianapolis, IN). Two µl of the reaction
product was used as an insert for TA ligation into pCRII (Invitrogen,
Carlsbad, CA) vector. Positive clones were analyzed by restriction analysis
and verified by sequencing the entire insert.
Preparation of Digoxigenin-Labeled Mouse Nephrin Riboprobe
Mouse nephrin antisense and sense riboprobes were prepared and labeled by
in vitro transcription using digoxigenin-labeled uridine triphosphate
(Boehringer Mannheim Biochemica, Mannheim, Germany) as described previously
(11).
A 2400-bp fragment of nephrin cDNA (positions 1154 to 3546) was subcloned into the EcoRI site of pCRII (Invitrogen BV, Groningen, The Netherlands) between SP6 and T7 promoters. After in vitro transcription, riboprobes were cut to approximately 300-bp fragments by alkaline hydrolysis and then used as probes (12).
Nonisotopic In Situ Hybridization
In situ hybridization was performed as described previously
(11). Briefly, sections of
Dubosq-Brazil-fixed, paraffin-embedded renal tissue were heat-fixed (65°C,
30 min) and deparaffinized. After permeabilization with proteinase K (40
µg/ml; Sigma-Aldrich, Milan, Italy), sections were hybridized with the RNA
probes at the final concentration of 0.1 to 0.5 ng/µl in 2 x SSC, 10%
dextran sulfate, 1 x Denhardt's solution, 20 mM Vanadyl Ribonucleoside
Complex (Life Technologies), and 0.1 M sodium phosphate and incubated
overnight in a moist chamber at 45°C. After being washed in 0.2 x
SSC and blocked with a buffer-blocking solution (50 mg/ml skimmed dried milk,
150 mM NaCl in 100 mM Tris HCl [pH 7.8]) at room temperature for 30 min, the
sections were incubated with antidigoxigenin antibody conjugated with alkaline
phosphatase (Boehringer Mannheim Biochemica) at the dilution of 1:1000 for 45
min at 37°C. Colorimetric detection with nitro blue tetrazolium salt and
5-bromo-4-chloro-3-indolyl phosphate (Boehringer Mannheim Biochemica) was then
performed, and the sections were mounted in 60% glycerol and examined by light
microscopy. The negative control included a hybridization step with the sense
probe. Thirty glomeruli for each rat were observed on average by a scientist
who was unaware of the nature of the experimental groups. The positivity of
nephrin mRNA was scored as follows: 0, no staining; 1, weak staining; 2,
staining of moderate intensity; 3, strong staining.
Preparation of Anti-Human Nephrin Antibody
The antigen used for generation of the rabbit anti-human nephrin polyclonal
antibody was purified in the following manner. PCR primers were designed for
amplification of human nephrin nucleotides 2000 to 2769, which correspond to
amino acids 668 to 923 of the extracellular domain, and subsequent cloning
into the bacterial expression vector pET-22b (Novagen, Madison, WI). The
resulting clone carries an in-frame, carboxy-terminal His
(6) tag for purification. After
purification on Ni-NTA agarose resin (Qiagen, Studio City, CA), the protein
was dialyzed to phosphate-buffered saline (PBS), quantified, and used to
immunize New Zealand white rabbits (Taconic, Germantown, NY). Rabbits received
a subcutaneous injection of 250 µg of purified antigen and were boosted at
3-wk intervals. Three wk after the third immunization, the animals were
killed, blood was harvested, and serum was then separated from the plasma for
use in immunohistochemical assays. Specificity of the antiserum was checked in
human nephrin transfected or untransfected HEK 293 cells. In Western blot
analysis, the antibody was detected to bind a 185- to 200-kD band obtained
from cell membrane preparation from transfected but not untransfected
cells.
Immunohistochemical Analysis
Localization of nephrin by immunohistochemistry was performed using a
rabbit anti-human nephrin antiserum raised against the extra-cellular domain
of human nephrin (amino acid residues 668 to 923, Ig repeat domains 7 and 8).
For ZO-1 immunoperoxidase, an affinity-purified rabbit anti-human ZO-1
antiserum (Zymed Laboratories Inc., San Francisco, CA) was used.
Dubosq-Brazil-fixed and paraffin-embedded sections were deparaffinized,
rehydrated, and incubated for 30 min with 0.3% H2O2 in
methanol to quench endogenous peroxidase. Tissue was permeabilized in 0.1%
Triton X-100 in PBS 0.01 M (pH 7.2) for 30 min, and aspecificities were
blocked by 30-min incubation with normal goat serum (Vector Laboratories,
Burlingame, CA). All of the above steps were carried out at room temperature.
Slides were then incubated overnight at 4°C in a moist chamber with the
primary antibody (anti-nephrin antiserum preabsorbed overnight with normal rat
serum and diluted 1:200 in PBS azide) followed by the secondary antibody
(biotinylated goat anti-rabbit IgG, Vector Laboratories),
avidin-biotin-peroxidase complex (ABC) solution, and finally developed with
diaminobenzidine. The sections were then counterstained with Harris
hematoxylin (Biooptica, Milan, Italy). Negative controls were obtained by
omitting the primary antibody on a second section present on all of the
slides. An average of 30 glomeruli for each rat were observed by a scientist
who was unacquainted to the nature of the experimental groups. Glomerular
positivity for nephrin was graded on a scale of 0 to 3 (0, no staining; 1,
weak staining; 2, staining of moderate intensity; 3, strong staining).
Statistical Analyses
Data are expressed as mean ± SD. Data were analyzed using the
nonparametric Kruskal-Wallis test for multiple comparisons. The statistical
significance level was defined as P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Situ Hybridization for Nephrin mRNA
The expression of nephrin mRNA was evaluated in rat kidney samples by
in situ hybridization using alkaline-digested riboprobes and reported
in Figure 1. An intense
expression signal of nephrin mRNA was detected in glomeruli of control animals
mainly restricted to podocytes. No specific signals were obtained with the
sense control probe (Figure 1).
A mild staining was observed occasionally in a few tubuli. This is not
surprising in view of the fact that similar findings were also reported in
figures of previously published papers
(13,14).
In control rats, nephrin staining in glomeruli was comparable at day 7 and at
2 and 4 mo of age and decreased at 8 mo. Animals with PHN showed a reduction
of glomerular mRNA for nephrin already at day 7
(Figure 2), when
glomerulosclerosis and tubulointerstitial damage were not yet present
(Table 1), which progressively
and remarkably decreased with time as shown by very consistent findings at
month 8 (Figure 1). Lisinopril
markedly prevented the decrease in nephrin mRNA transcript in podocytes
(Figure 1). To assess whether
the favorable effect of ACE inhibition was actually due to a reduced activity
of AII, we evaluated the action of an AII receptor blocker, L-158,809, on
renal nephrin gene expression. L-158,809 was as effective as the ACE inhibitor
in preventing the decrease of nephrin mRNA transcript; almost all glomeruli
showed a positive staining for nephrin
(Figure 1).
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The intensity of the staining for nephrin mRNA was calculated by a semiquantitative score and reported in Figure 2. Intensity of the staining was comparable in control rats studied at day 7 and at 2 and 4 mo and was reduced by 33% in control rats at month 8 (Figure 2). In PHN rats, intensity of the nephrin transcript was already reduced by 17% at day 7 and further decreased by 67% at month 4 in respect to controls. At month 8, nephrin mRNA in PHN animals was virtually absent with only very few glomeruli revealing a very faint nephrin staining (Figure 2). Treatment of PHN rats with the ACE inhibitor or the AII receptor blocker induced a normalization of the staining for nephrin that reached levels comparable to that of control rats (Figure 2).
Localization of Nephrin in the Kidney from PHN Rats
Consistent with patterns of nephrin mRNA expression, immunohistochemistry
of normal rat kidneys with the antibody against the extracellular nephrin
portion showed a prominent glomerular localization. A linear dotted line of
reactivity giving a preferentially epithelial-like staining pattern could be
observed. Immunohistochemical staining of nephrin was comparable in control
rats at day 7 and at 2 and 4 mo and tended to decrease at 8 mo. PHN rats
studied at day 7 had a decrease in nephrin staining that progressively lowered
with time at 4 and 8 mo (Figure
3). A semiquantitative score of glomerular positivity for the
light microscopy immunostaining was calculated for each animal and reported in
Figure 4. At day 7 and at 2 and
4 mo, almost all glomeruli were positive for nephrin, whereas a reduction of
the positivity score was observed at 8 mo
(Figure 4). Untreated PHN
animals exhibited a significant (P < 0.05) reduction in the
semiquantitative score over controls already at day 7, which further
exacerbated at 4 mo. Nephrin reactivity dramatically decreased in podocytes of
PHN animals at 8 mo to levels that were significantly lower than those of
control animals at the corresponding time (P < 0.05). Conversely,
the administration of the ACE inhibitor markedly prevented the decrease of
nephrin expression in PHN animals. The score of positivity in PHN animals that
received the ACE inhibitor was remarkably higher than that of control animals
of the corresponding age (month 8) and fairly comparable to the score of
control animals studied at day 7 and at 2 and 4 mo
(Figure 4). Consistent with the
data obtained in these latter groups, PHN animals that were given the ACE
inhibitor had all glomeruli positive for nephrin protein expression. The same
trend was observed with the AII receptor blocker with a positivity score very
comparable to that of control animals and almost all glomeruli positively
stained (Figure 4), strongly
indicating a role for AII in inducing changes of nephrin protein expression in
the glomerulus in PHN animals.
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To evaluate whether reduction of nephrin expression was specific, we performed the immunostaining of another component of the slit diaphragm, ZO-1, at an early and late stage of the disease of PHN. At day 7, expression of ZO-1 was fairly comparable to values observed in controls (score, 2.9 ± 0.1 versus 2.9 ± 0.1). At 8 mo, control animals had a slightly lower score of positivity that was on average 2.1 ± 0.6. PHN rats studied at the same time, when renal damage was very severe, revealed a lower staining for ZO-1 accounting 1.8 ± 0.2, a value numerically but not statistically different from that of controls. The appreciable difference between nephrin and ZO-1 reduction in PHN rats at month 8 (79 versus 15%) suggests that changes in nephrin mRNA and protein were specific and not part of a generalized phenomenon.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here we report that in an accelerated model of experimental nephrosis reminiscent of human membranous nephropathy, the glomerular expression of nephrin gene decreased over time in parallel to the development of proteinuria and the subsequent renal damage. Concomitant with the time-dependent reduction in gene expression, nephrin formation was also impaired. This phenomenon was reasonably specific for nephrin because the expression of another protein of the slit diaphragm ZO-1 was not affected substantially. Downregulation of glomerular nephrin production as early as 7 d after the induction of the disease indicates a pathogenetic role in permeability changes. A time-dependent reduction of nephrin mRNA together with a decreased staining of the cortical kidney with an antibody to the intracellular nephrin domain was observed previously in glomeruli from rats with puromycin amino nucleoside nephrosis (17). Altered expression of nephrin mRNA was reported also in nephrotic syndrome patients with idiopathic membranous glomerulonephritis or minimal change nephropathy whose glomerular nephrin mRNA was reduced in comparison with the transcript of normal, nonproteinuric glomeruli (18).
PHN was commonly used in the past to understand the mechanisms underlying the disease pathology of the human counterpart membranous nephropathy. Changes in the ultrastructure of the glomerular capillary wall components including a decreased frequency of the filtration slits and an increased GBM thickness has been described in PHN rats (3). Theoretical analysis of morphometric parameters allowed an appreciation of a reduction of hydraulic permeability to water and albumin of the GBM as well as the epithelial layer, which have been considered as possibly being responsible for the filtration power in this model (3). Treatment with lisinopril was associated with a preserved frequency of epithelial slits in the face of abnormal GBM thickness. Lisinopril also prevented, at least in part, GFR decline and ameliorated the hydraulic permeability of the GBM (3). The above study already suggested that the clue for the effect of ACE inhibitor of preserving membrane filtration barrier must reside on slit diaphragm structure or function. Our previous studies also documented significant changes in the distribution of the tight junction protein occludens-1 (ZO-1)—which plays a crucial role in maintaining the functional properties of the epithelial slit diaphragm—in rats genetically programmed to develop spontaneous proteinuria with age (19). This alteration was not accompanied by any apparent ultrastructural change of the foot processes and the epithelial slit diaphragms and was prevented by ACE inhibition that also effectively reduced protein excretion rate in these animals (19). Interestingly, in this model, amelioration of glomerular ultrafiltration coefficient induced by the ACE inhibitor was not a consequence of changes in filtering surface area but rather reflects an increase in membrane hydraulic permeability properties (19). In the present study, we also found that blocking the biologic effects of AII either with an ACE inhibitor or with an AII receptor antagonist fully prevented nephrin rearrangement observed in untreated animals with a severe form of PHN. The resulting well-known effect of these drugs of limiting urinary proteins in this and other animal models of progressive nephropathy therefore may depend on the capacity of AII blockers to preserve slit diaphragm functional integrity of which nephrin seems to represent a crucial aspect. Indirectly, these data imply a role for AII in modifying the size-selective properties of the glomerular capillary barrier. This is supported further by direct evidence from our laboratory and others' that continuous infusion of AII into the renal artery induces a progressive and significant increase in urinary protein excretion rate accompanied by enhanced fractional clearance of tracer molecules corresponding to the size of albumin as a function of intracellular signaling through the angiotensin II type 1 receptor (5,20).
Potential AII-independent mechanisms of preservation of nephrin expression by AII blockade also could be considered. Those include modification in the development of immune deposits and changes in epithelial cell morphology. Concerning the former, a significant reduction in the number and the extension of electron-dense deposits within the GBM was observed previously in PHN rats given lisinopril in respect to untreated animals (3). As far as podocyte morphology, some very recent data obtained in the same model by Yuan et al. (21) found changes in nephrin distribution before the appearance of proteinuria with subsequent disruption of nephrin staining in the more proteinuric phase. Changes in nephrin corresponded to the deposition of C5b-9, suggesting the possibility that complement-mediated podocyte injury alters the slit diaphragm as to eventually account for proteinuria.
The effect of ACE inhibitors and AII receptor antagonists on nephrin biosynthesis is an unprecedented finding. Currently, the mechanism responsible for downregulation of nephrin mRNA and protein in conditions of excessive renal AII is unknown. Whatever the mechanism by which AII blockade maintains nephrin expression, either by directly reducing AII or by preserving podocyte and/or foot process architecture, the present study discloses a novel pathway linking ACE inhibitors and AII receptor antagonist to renoprotection. The time-dependent downregulation of the nephrin seems to be the molecular equivalent of the recruitment of large unselective pores responsible for proteinuria in this and other model of progressive nephropathies. Recent findings that the slit area can be modified by enhancing intraglomerular pressure offer a molecular explanation for the old observation that glomerular hemodynamic changes translate into opening of large glomerular pores and protein trafficking and for the protective effect of limiting AII on both parameters.
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Acknowledgments |
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The authors gratefully acknowledge Daniela Corna for technical assistance and Dr. Mauro Abbate for help in the immunohistology experiments and antibody purification. E.G. is a recipient of a fellowship of Associazione Ricerca Malattie Rare (ARMR).
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 A. Koziell, V. Grech, S. Hussain, G. Lee, U. Lenkkeri, K. Tryggvason, and P. Scambler Genotype/phenotype correlations of NPHS1 and NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in glomerular filtration Hum. Mol. Genet., February 1, 2002; 11(4): 379 - 388. [Abstract] [Full Text] [PDF]
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