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Regular Articles |
From the Department of Clinical Pathology,*
University
of Vienna, Vienna, Austria, and the Institut für
Diabetesforschung,
Munich, Germany
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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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There are several instructive experimental animal models for MCN/FSGS that show features similar to the human disease. One well studied example relies on intoxication of rats with puromycin aminonucleoside.5 Podocytes are very sensitive to this toxic antibiotic for unknown reasons and respond promptly with foot process retraction6 and proteinuria at the nephrotic level. In this and several other experimental glomerular diseases overproduction of reactive oxygen species (ROS) and local accumulation of lipid peroxidation (LPO) adducts were identified as prime causes for glomerular damage7 and interventional therapies with specific scavengers drastically reduced foot process flattening and proteinuria.8 Although these model diseases require exogenous intoxication and cause complex reactions of podocytes, similar symptoms of disease were found to occur spontaneously in mice in which the gene Mpv17 was inactivated by retroviral insertion (Mpv17-/- mice).9 Here we have examined the possibility that in this system foot process flattening and proteinuria are also caused by excessive production of ROS and LPO adducts in glomeruli.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hanks' balanced salt solution, luminol (5-amino-2,3-dihydro-1,4-pthalaiznedione), probucol, and dimethylthiourea (DMTU) were obtained from Sigma Chemical Corp. (St. Louis, MO) and methylprednisolon from Upjohn Corp. (Kalamazoo, MI). Rabbit anti-Mpv17 IgG was prepared and characterized as previously described.10 Tetramethylrhodamine isothiocyanate-labeled goat anti-rabbit IgG was from DAKO (Copenhagen, Denmark).
Mpv17-/- Mice
Homozygous Mpv17-/- mice were generated by retroviral insertion as described elsewhere.9 As these original Mpv17-/- mice showed reduced breeding capacity and died from renal failure within a few months after birth, in this study we used a strain selected for extended survival (Weiher, unpublished observations). This strain showed proteinuria and histopathological features of FSGS, as did the original Mpv17-/- mice, but failed to develop interstitial fibrosis. Development of glomerular disease was documented by routine histology in paraffin sections and by standard electron microscopy. As controls, age-matched BALB/c mice were used. Use of experimental mice was authorized by the Austrian Ministry of Science and Traffic.
Scavenger Treatment
One group of Mpv17-/- mice was treated with DMTU administered by osmotic pumps11 (Alzet 2002, 200 µl; 0.5 µl/hour, Alza Corp., Palo Alto, CA) filled with 312 mg/ml DMTU in phosphate-buffered saline (PBS),8 or with PBS alone in controls. Pumps were implanted subcutaneously into the back between the scapulae of anesthetized mice at postnatal day 29 and were active for exactly 14 days. The pump's weights were determined before implantation and after explantation to calculate the exact amounts of DMTU delivered. The average dose of DMTU administered was 15 µg per kilogram of body weight per day. Treated and control animals were sacrificed as indicated in the respective figures.
Probucol was administered to Mpv17-/- mice by feeding standard chow pellets containing probucol (1% w/w, Altromin, Lage, Germany). Experimental animals were exposed to probucol from early in their mothers' pregnancy until their sacrifice. Five Mpv17-/- mice were injected intraperitoneally with methylprednisolon (30 mg/kg) at postnatal day 57 and proteinuria was recorded for 10 days.
Proteinuria
Urine was collected from Mpv17-/- and control BALB/c mice and proteinuria was determined by Pierce Coomassie Blue and albumin assays conducted according to the manufacturer's instructions.
Quantitative Electron Microscopy
Development of podocytes was recorded by quantitative electron microscopy, as described.12 Kidney cortex samples were fixed in 2.5% glutaraldehyde/4% paraformaldehyde in 100 mmol/L Na cacodylate buffer, followed by OsO4 in the same buffer. After rinsing in distilled water, the samples were incubated in 0.5% uranyl acetate and embedded in epoxy resin. The number of foot processes per micrometer of glomerular basement membrane was determined from electron micrographs of capillary loops of at least 3 different glomeruli per kidney.
Histochemistry and Immunofluorescence Microscopy
The glomerular polyanion was visualized on paraffin sections of treated and untreated Mpv17-/- and BALB/c control kidneys by a colloidal iron procedure, followed by a Prussian blue reaction.13 Cryostat sections (3 µm) of unfixed Mpv17-/- mouse kidneys were incubated with rabbit anti-Mpv17 IgG (1 µg/ml), followed by tetramethylrhodamine isothiocyanate-conjugated goat anti-rabbit IgG.
Detection of Oxygen Radicals by the Luminol Method
Forty-day-old proteinuric Mpv17-/- and control BALB/c mice were ether anesthetized and perfused with Hanks' buffered salt solution via the left cardiac ventricle. Isolation of glomeruli was performed by sequential sieving and differential centrifugation. The purity of the fractions was >90% as determined by direct microscopy. Isolated glomeruli were incubated in Hanks' buffered salt solution for 15 minutes at 37°C, and the production of oxygen radicals was determined by measuring the peak of chemiluminiscence induced with 5-amino-2,3-dihydro-1,4-pthalaiznedione (luminol) at a final concentration of 3 x 10-5 M in a Beckman LS6500 liquid scintillation counter (Beckman Instruments, Fullerton, CA) at 25°C.14
Detection of Lipid Peroxidation Adducts
Kidneys of Mpv17-/- and of control BALB/c mice were perfused via the left cardiac ventricle with ice-cold PBS. After decapsulation kidneys were weighted, snap frozen in liquid N2, and homogenized in a microdismembrator. Tris-HCl buffer (pH 7.4) was added to the homogenates (10 ml/g) and the tissue suspension was centrifuged at 3000 x g for 10 minutes. The concentrations of malondialdehyde (MDA) and 4-hydroxynonenal per milligram of kidney protein were determined by the LPO test kit (Bioxytech LPO-586, Oxis International, Portland, OR). In this test different substrates were used to indicate the concentration of MDA alone or of MDA plus 4-hydroxynonenal combined according to the manufacturer's instructions.
Statistical Methods
Data are expressed as mean and standard deviations (SD) of the respective groups or time points, as indicated in the figure legends. Differences between control and Mpv17-/- mice as well as the effects of treatment on proteinuria and the number of foot processes were analyzed by the variance test. Differences between control and Mpv17-/- mice with respect to the generation of oxygen radical formation and lipid peroxidation products in isolated glomeruli and kidney homogenates, respectively, were analyzed by the unpaired t-test. A P value <0.05 was regarded as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histopathological changes of glomeruli of Mpv17-/- mice
were examined 50 and 180 days after birth, and several
morphological features were found that closely resemble human
FSGS.1
At 50 days, glomeruli showed slight
enlargement and segmental mesangial sclerosis (Figure 1A)
, and at 180 days hyaline and scarred
segmental lesions were also observed (Figure 1, B–D)
. Interstitial
inflammation and fibrosis were not present. By electron microscopy
almost complete flattening of foot processes was found, as shown
previously.9
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Diffuse to fine granular staining of podocytes was observed
on cryostat sections of normal mouse kidney (Figure 2A)
by indirect immunofluorescence with
affinity purified antibody raised against the recombinant Mpv17
protein. In proximal tubular epithelial cells a coarse granular pattern
was obtained, presumably corresponding to peroxisomes (Figure 2C)
. By
contrast, both glomerular and tubular staining were abolished in
the Mpv17-/- mouse (Figure 2, B and D)
. These data are in agreement
with previous biochemical data, confirming lack of the Mpv17 gene
expression in Mpv17-/- mice.9,10
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When the overall amount of negatively charged groups in
glomeruli was determined by cationic colloidal iron cytochemistry,
strong labeling was observed in controls and Mpv17-/- mice treated
with probucol or DMTU (Figure 2E)
, but only traces were detected in
proteinuric Mpv17-/- mice (Figure 2F)
. Labeling of endothelia of
interstitial blood vessels was similar in all groups.
Proteinuria in Mpv17-/- Mice Is Selective
Total proteinuria was determined by the Coomassie blue assay, and albumiuria with a mouse albumin-specific assay. It was observed that < 95% of the proteinuria was accounted for by urinary albumin excretion (data not shown).
Isolated Glomeruli of Mpv17-/- Mice Produce Excessive ROS and Contain LPO Adducts
ROS production by equal numbers of isolated glomeruli of
BALB/c-controls and Mpv17-/- mice was measured by the luminol assay
that provides an overall estimate on a wide variety of ROS. A
statistically significant twofold increase in ROS production/glomerulus
over controls was observed in Mpv17-/- mice (Figure 3A)
.
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). Similar results were obtained for MDA plus
4-hydroxy-2(E)-nonenal, another LPO adduct (data not shown). Podocyte Damage and Proteinuria Develop in Mpv17-/- Mice after Maturation of Glomeruli
For the targeting of a pharmacological intervention it was
necessary to determine the optimal time point for treatment at the
onset of development of the glomerular lesions. Therefore the
maturation of podocytes was recorded by quantitation from conversion of
flattened podocytes at birth to full development of the arborized foot
processes by quantitative electron microscopy. Subcapsular glomeruli
were analyzed to minimize the sampling error. In normal BALB/c mice,
full maturation of podocytes was observed stereotypically at days
30–32 postpartum. The same time schedule of podocyte maturation was
also observed for Mpv17-/- mice. However, immediately after reaching
full expansion, their foot processes started to flatten again until
neonatal levels of spreading were restored on days 33–35 postpartum
(Figure 4)
that remained for the rest of
the animal's life.
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. Oxygen Radical Scavengers Prevent Glomerular Damage in Mpv17-/- Mice
The potent oxygen radical scavenger DMTU was infused continuously
at a constant rate via an implanted minipump11
for 2
weeks. The start of the infusion was set at day 29 postpartum to cover
the entire period of foot process flattening after initial maturation.
The major finding was that the mature foot process morphology was
preserved, precisely as long as the pump was active and the level of
DMTU was maintained (Figure 5)
.
Immediately after exhaustion of the DMTU reservoir the foot processes
rapidly converted into flat epithelial sheets in 1–3 days (Figure 6A)
. Within the follow-up of 2 weeks,
proteinuria redeveloped only slowly (Figure 6B)
. The results obtained
in Mpv17-/- mice were controlled by a group of Mpv17-/- mice with a
minipump containing PBS. In these controls foot process flattening
developed identically to that in unmanipulated mice (Figure 5)
.
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Proteinuric Mpv17-/- mice treated with a single dose of methylprednisolon at postnatal day 57 failed to show any effect on proteinuria within 10 days of follow-up and podocytes remained flat (data not shown).
The Lipid Peroxidation Scavenger Probucol Prevents Glomerular Damage in Mpv17-/- Mice
To saturate experimental animals with the lipophilic drug
probucol, breeding pairs of Mpv17-/- mice were kept on a diet
containing probucol for 2 months, and their offspring were also fed
chow containing probucol after birth. Development of foot processes was
followed by quantitative electron microscopy and urine protein
concentration was monitored. When compared to control BALB/c mice, no
differences in the time course of postnatal development of foot
processes were found and foot process morphology was completely
preserved, even at time points when their architecture was completely
lost in untreated Mpv17-/- mice (Figure 7A)
. Proteinuria was completely absent
(Figure 7B)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ROS were recently found to mediate glomerular damage in several experimental models of renal disease7 and we have therefore investigated in this study their role in the glomerular disease of homozygous mice lacking functional Mpv17 genes. The results of this study provide evidence that increased production and/or reduced degradation of extracellularly released ROS within glomeruli of Mpv17-/- mice is causally related to the glomerular disease observed in these animals. This interpretation rests primarily on direct demonstration of enhanced ROS production by isolated glomeruli of Mpv17-/- mice, and on interventional therapies with specific ROS scavenging drugs.
The precise biochemical mechanisms of increased glomerular ROS production in MPV17-/- mice are unclear. Using hyroethidine that is converted into a fluorescent dye by superoxide radicals and other ROS species, we previously reported that overexpression of the Mpv17 protein resulted in accumulation of ROS within tissue culture cells and consequently proposed that knockout of this protein should reduce ROS production.10 However, the use of fluorescent or luminescent compounds of low molecular weight to detect ROS is not unanimously accepted19 and scavengers of high molecular weight, such as superoxide dismutase and catalase, pose the problem of access to ROS in glomeruli in vitro and in vivo. One major point of criticism is that ROS could be self-generated by the detecting dyes19 but, by contrast, the specificity of luminol for detection of ROS was recently validated.20 To clarify this controversial issue of detectors biasing ROS determinations in Mpv17-/- cells, we have performed in vitro electron spin resonance experiments that directly revealed a significantly increased release of superoxide anion into the culture media by Mpv17-deficient cells when compared with Mpv17-proficient cells (Wagner and Weiher, manuscript in preparation). This finding is compatible with the concept of increased release and extracellular accumulation of ROS in glomeruli of Mpv17-/- mice, but it remains to be determined why pathology develops preferentially in glomeruli and is not more widespread.
In vitro studies have provided evidence that ROS cause direct, profound effects on the integrity and composition of basal membrane matrix proteins.21 ROS produced by the Fenton reaction were shown to disintegrate and fragment EHS sarcoma matrix in vitro in a dose-dependent fashion, with entactin and laminin being most sensitive to detachment and degradation and type IV collagen most resistant. It remains to be determined whether similar events also occur in vivo in Mpv17-/- mice. Heparan sulfate proteoglycan extracted from glomerular basement membrane (GBM) was incubated in vitro with ROS generated by xanthine-xanthine oxidase,22 resulting in specific degradation of glucosaminoglycan side chains and reduced charge density of the proteoglycan. Thus, ROS-mediated destruction of glucosaminoglycans could contribute to loss of the "glomerular polyanion" and selective proteinuria observed in Mpv17-/- mice, because reduction of electronegatively charged groups is known to increase GBM permeability selectively for anionic molecules such as albumin.23
There is increasing evidence that flattening of podocytes could be not only a consequence but also the cause of increased glomerular permeability. Multiple and diverse stimuli act on podocytes to induce shape changes. They include toxic reactions, such as puromycin aminonucleoside and adriamycine, decrease in podocyte surface charge by neuraminidase digestion,24 and compensation with the polycations protamine sulfate and L-polylysine.12 Other means leading to the same effect are modification of the GBM composition, eg, by genetic knockout of s-laminin,25 or in situ immune complex formation with epitope-specific antibodies to podocyte membrane proteins such as podoplanin,26,27 aminopeptidase A,28 and p51,29 and with the antigen of Heymann nephritis, gp330/megalin.30 However, at present one can only speculate about mechanisms of ROS-induced foot process deformation in the Mpv17-/- mouse. It is possible that chemical ROS-induced modifications of matrix or adhesion molecules play a role, but actions of ROS-activated transcription factors cannot be excluded. Intriguingly, postnatal development of podocytes follows the same pattern of maturation in Mpv17-/- mice as in controls. Assuming that innate overexpression of ROS was established at birth, it is possible that scavengers yet unidentified are produced within glomeruli or resorbed from the mother's milk, or that antioxidative compounds are expressed only during the postnatal period and decrease later.
In addition to the glomerulosclerotic phenotype Mpv17-/- mice showed pathological changes in their inner ear, reminiscent of Alport's syndrome.31 Indeed, as in Alport's syndrome, both the glomerular and the cochlear basement membranes are affected and recent results indicate that the matrix metalloprotease II (MMP-2) plays a key role in progression of this cochlear disease.18 It is also possible that increased release of MMP-2 is involved in glomerular damage; however, the results of this study clearly indicate that this could be only a consequence of ROS-induced damage, as selective ROS scavenging globally reduced glomerular lesions.
Molecular mechanisms of GBM damage and proteinuria are relatively well understood in Heymann nephritis, a model of human membranous nephropathy.15 A detailed analysis of its pathogenesis of proteinuria revealed a chain of events that include intraglomerular overproduction of ROS, generation of lipid peroxidation adducts in the GBM, and crosslinking of Type IV collagen via its NC-1 domains. Consequently, therapy with the lipid peroxidation scavenger probucol efficiently reduced proteinuria,15 and probucol was recently identified as effective antiproteinuric agent in human patients with membranous nephropathy. (Meyer G, Haas M, Wirnsberger G, Holzer H, Ratschek M, Neyer U, Neuweiler R, Kramar R, Schneider B, Breiteneder S, Regele H, Hörl W, Kerjaschki D, unpublished manuscript). Intraglomerular LPO is also clearly involved in the glomerular disease of Mpv17-/- mice, because LPO adducts were found in high concentrations in isolated glomeruli of Mpv17-/- mice and probucol completely prevented the disease when it was fed to Mpv17-/- mice from birth. The possibility that in this setting the major LPO product MDA32 was generated as a byproduct of increased glomerular prostaglandin synthesis rather than as a consequence of LPO is unlikely, because its formation was reduced by probucol, which has no influence on prostaglandin synthesis. It remains to be shown whether probucol is also effective in interventional therapy and reverses the glomerular changes in advanced stages of Mpv17-/- disease.
Collectively, the results of this study provide evidence for the central involvement of ROS and LPO in the pathogenesis of the Mpv17-/- glomerulopathy. This mouse disease resembles human FSGS not only in its morphology and proteinuria, but also in its resistance to a bolus therapy with a high dose of steroids. Thus, it will be of some interest to determine the pathogenic role of glomerular ROS production and the effect of antioxidative therapy in this human disease.
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Footnotes |
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Supported by Sonderforschungsbereich 05, Project 007 from the Österreichischen Fonds zur Förderung der Wissenschaftlichen Forschung and the EC Concerted Action Contract no. BMH4–98-3631 (to D.K.).
Dr. Binder's current address: Division of Endocrinology and Metabolism, Department of Internal Medicine, University of California, San Diego, San Diego, California.
Accepted for publication January 22, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-linked sialic acid from the glomerular filtration barrier results in podocyte charge alteration and glomerular injury. Lab Invest 1996, 74:907-920[Medline]
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