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8 Integrin Chain Affords Mechanical Stability to the Glomerular Capillary Tuft in Hypertensive Glomerular Disease
From the Medizinische Klinik IV,*
Universität Erlangen-Nürnberg, Erlangen, Germany; and the Friedrich-Miescher-Institut,
Basel, Switzerland
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Abstract |
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8 integrin chain is expressed in
glomerular mesangial cells. The 8 integrin plays a role in early
nephrogenesis but its functional role in the adult kidney is unknown.
We tested the hypothesis that 8 integrin-mediated cell-matrix
interactions are important to maintain the integrity of the glomerulus
in arterial hypertension. Desoxycorticosterone (DOCA)-salt hypertension
was induced in mice homozygous for a deletion of the 8 integrin
chain and wild-type mice. Blood pressure, albumin
excretion, total renal mass, and glomerular filtration
in DOCA-treated 8-deficient mice were comparable to DOCA-treated
wild types. DOCA-treated wild types showed increased glomerular
immunostaining for 8 integrin compared to salt-loaded and untreated
controls, whereas the glomeruli of 8-deficient mice always
stained negative. Morphometric studies revealed similar degrees of
glomerulosclerosis in DOCA-treated 8-deficient and DOCA-treated
wild-type mice. However, DOCA-treated 8-deficient mice had a
higher score of capillary widening (mesangiolysis) than DOCA-treated
wild-type mice, which was confirmed in two additional wild-type
strains. Moreover, in DOCA-treated 8-deficient mice,
glomerular fibrin deposits were more frequent than in DOCA-treated wild
types. The results show that lack of 8 is associated with increased
susceptibility to glomerular capillary destruction in DOCA salt
hypertension, whereas it does not seem to play a major role in
the development of fibrosis or glomerulosclerosis. Our findings
indicate that mesangial 8 integrin contributes to maintain the
integrity of the glomerular capillary tuft during mechanical
stress, eg, in hypertension.
Integrins are heterodimers consisting of an and a ß subunit. The
8 integrin subunit was shown to be a partner for ß1.3
8ß1 binds the RGD site of fibronectin, vitronectin, tenascin
C,4
osteopontin,5
and the recently described
nephronectin.6
In the kidney, we and
others7,8
showed expression of 8 integrin in smooth
muscle cells of renal arterioles and glomerular mesangial cells.
Furthermore, glomerular 8 integrin was increased in a transient
model of mesangioproliferative glomerulonephritis during matrix
expansion.7
However, the function of 8 integrin in the
mesangium of healthy and diseased glomeruli is still unclear.
During development, 8 integrin is induced in the mesenchyme
surrounding the branching ureter tips.9
Mice deficient for
the 8 integrin chain have severe defects in ureteral branching,
frequently leading to agenesis or dysgenesis of the kidney. Many of
these mice die shortly after birth. There are apparently no
abnormalities in later stages of kidney development; however, surviving
animals show unilateral kidneys or only small kidneys with normal
appearing glomeruli.9
Thus, 8 does not seem to be
essential for glomerulogenesis.
We analyzed the role of 8 in the maintenance of glomerular structure
and function in the face of increased mechanical strain. Glomerular
hypertension was induced in uninephrectomized wild-type and 8
integrin-deficient mice by administration of
desoxycorticosterone-acetate (DOCA) and 1% NaCl as drinking water.
This model was chosen because here the glomeruli of the kidney are
uniquely exposed to high blood pressure.10
In view of the
prominent glomerular abundance of 8 integrin, we hypothesized that a
lack of 8 would aggravate glomerular injury, leading to more severe
morphological alterations.
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Materials and Methods |
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Mice were housed in a room maintained at 22 ± 2°C, exposed to a 12-hour dark/light cycle. The animals were allowed unlimited access to chow (no.1320; Altromin, Lage, Germany) and tap water (or 1% saline, see below). All procedures performed on animals were done in accordance with guidelines of the American Physiological Society and were approved by the local government authorities (Regierung von Mittelfranken, AZ no. 621-2531.31-1/01).
Male homozygous 8 integrin-deficient mice generally develop only one
kidney or two smaller kidneys, leading to a reduced total renal mass.
In this study, only 8-deficient mice with two smaller kidneys were
used. 8-deficient mice were not backcrossed into C57/BL6 but
maintained as a homozygous line derived from the first generation of
homozygotes.9
Age- and weight-matched male 129 mice
(Charles River, Sulzfeld, Germany) were used as the primary wild-type
controls. Being aware that 129 mice are not the ideal wild-type control
for 8-deficient mice, we additionally investigated two more
wild-type strains: C57/BL6 mice and 129xC57/BL6 intercrosses.
At an average weight of 16 g to 18 g, wild-type mice
underwent right unilateral nephrectomy to adjust for the reduced kidney
mass in 8-deficient mice. 8-deficient mice were not
nephrectomized. After 2 weeks of recovery, 21-day-release DOCA pellets
containing 50 mg of DOCA (Innovative Research of America, Sarasota, FL)
were implanted subcutaneously by incision of the right flank under
light ether anesthesia. Control animals were sham operated. After 21
days they received a replacement pellet. All animals (DOCA and control
groups) received isotonic saline (10 g NaCl/L) for drinking for 6
weeks, starting with the first day of DOCA treatment. The animals were
then followed by weekly measurements of weight and systolic blood
pressure (Visitech Systems, Apex, NC).
After 6 weeks of treatment, the mice were put into metabolic cages, and
urine was collected for 24 hours for measurement of albuminuria. The
urinary albumin content was measured by an enzyme immunoassay kit
(EIA) (CellTrend, Luckenwalde, Germany). Finally, animals were
sacrificed (6 DOCA-treated 129 mice, 10 DOCA-treated 8-deficient
mice, 6 129 control mice, and 10 8-deficient controls). At the day
of sacrifice, animals were equipped with a carotid artery catheter
under ketamine/xylazine anesthesia and intra-arterial blood pressure
was measured in conscious mice 4 hours after anesthesia. Animals were
sacrificed by dissecting the abdominal artery and bleeding in deep
ketamine/xylazine anesthesia.
After measuring kidney weight, the organs were decapsulated. Kidneys were put in methyl-Carnoy solution (60% methanol, 30% chloroform, and 10% glacial acetic acid) for fixation.
Renal Histology
After overnight fixation in methyl-Carnoy solution, tissues were dehydrated by bathing in increasing concentrations of methanol, followed by 100% isopropanol. After embedding in paraffin, 2-µm sections were cut with a Leitz SM 2000 R microtome (Leica Instruments, Nussloch, Germany). Before any staining procedure, sections were deparaffinized by bathing in xylol and rehydrated in decreasing concentrations of alcohol. For evaluation of general renal histology, kidney sections were periodic acid-Schiff (PAS) stained.
To quantify glomerulosclerosis a score of 0 to 4 was used as described before.11 A score of 0 indicated normal glomerulus, a score of 1 indicated mesangial expansion or sclerosis involving up to 25% of the glomerular tuft, a score of 2 indicated sclerosis 25 to 50%, a score of 3 indicated sclerosis 50 to 75% and/or segmental extracapillary fibrosis or proliferation, and a score of 4 indicated global sclerosis (>75%) or global extracapillary fibrosis or proliferation, or complete collapse of the glomerular tuft. To assess the extent of the formation of microaneurysms, capillary widening was scored. Similar to the score for sclerosis, a score 0 to 4 was applied. Score 0 indicated no capillary widening in the glomerular tuft, score 1 indicated a capillary widening involving up to 25% of the glomerular tuft, score 2 indicated a capillary widening 25 to 50%, score 3 indicated a capillary widening 50 to 75%, and a score of 4 indicated capillary widening involving >75% of the glomerular tuft.
Antibodies
The rabbit polyclonal antiserum to 8 integrin was used at a
dilution of 1:200 as described before.7
A monoclonal
antibody to smooth muscle actin (DAKO Diagnostika, Hamburg, Germany)
was used at a dilution of 1:50 to stain activated myofibroblasts. A
mouse monoclonal antibody to fibrin (American Diagnostica, Pfungstadt,
Germany) was used at a dilution of 1:1000 to stain fibrin exudates
after blocking of endogenous mouse IgG by a mouse-on-mouse kit
(MOM) (Linaris, Wertheim, Germany). Rabbit polyclonal
antibodies to fibronectin (Life Technologies, Eggenstein, Germany),
collagen I (Biogenesis, Poole, England), and collagen IV (Southern
Biotechnology Associates, Birmingham, AL) were used at
adilution 1:1000.
Immunohistochemistry
In deparaffinized kidney sections, endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 20 minutes at room temperature.
Sections were then layered with the primary antibody and incubated at 4°C overnight. After addition of the secondary antibody (dilution, 1:500; biotin-conjugated goat anti-rabbit IgG or rabbit anti-mouse IgG, all Dianova, Hamburg, Germany), the sections were incubated with avidin horseradish peroxidase complex and exposed to 0.1% diaminobenzidine tetrahydrochloride and 0.02% H2O2 as a source of peroxidase substrate. The Vectastain DAB kit (Vector Laboratories, Burlingame, CA) was used as a chromogen. Each slide was counterstained with hematoxylin. As a negative control, we used equimolar concentrations of preimmune rabbit or mouse IgG.
Expansion of interstitial collagen I was measured in a Leitz Aristoplan
microscope (Leica Instruments) by Metaview software (Visitron Systems,
Puchheim, Germany) in 10 nonoverlapping medium-power cortical views per
section excluding glomeruli and was expressed as percentage of stained
area per cross-section. Glomerular 8, fibronectin, and collagen IV
staining was measured by Metaview in every third glomerulus per
cross-section and was expressed as percentage of stained area per
glomerular tuft.
Analysis of Data
Two-way analysis of variance, followed by post hoc Newman-Keuls test with adjustment for multiple comparisons, was used to test significance of differences between groups. A P value <0.05 was considered significant. The procedures were performed using the Statistica software (StatSoft, Tulsa, OK). Values are displayed as means ± SEM.
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Results |
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8
Immunoreactivity
Wild-type mice had expanded and more intense mesangial staining
for 8 integrin when treated with DOCA as compared to salt-treated
controls (Figure 1)
. In wild-type
controls, 8 staining covered 5.0 ± 0.8% of glomerular area,
whereas in DOCA salt-treated wild types 17.7 ± 1.7% of the
glomerular tuft stained positive for 8. Another cell-type staining
with the 8 integrin antibody in the mouse kidney was the smooth
muscle cell of the renal vasculature. Treatment with DOCA salt
increased vascular smooth muscle staining for 8 (not shown). No
staining for 8 in renal interstitial fibroblasts was detected in
control animals. However, in some DOCA-treated animals 8
immunoreactivity was detected in clusters of renal interstitial
fibroblasts (not shown). All 8-deficient mice were negative for 8
immunoreactivity in all renal cells, confirming the lack of 8
expression in this mouse strain.
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8-Deficient and Wild-Type Mice Develop a Comparable Degree of
Hypertension and Hypertensive Nephrosclerosis
8-Deficient mice of the control group had a slightly higher
blood pressure than 129 wild-type controls. However, DOCA treatment led
to a significant and comparable increase in mean arterial blood
pressure both in 8-deficient and wild-type mice (Figure 2)
. DOCA-treated wild-type and
8-deficient mice developed hypertension-dependent alterations to a
similar degree, comprising a marked rise in relative weights of kidney
and left ventricle, a decrease of creatinine clearance, and augmented
albuminuria (Table 1)
. Dramatic
histological changes were detected in all DOCA-treated animals that
reached comparable values in wild-type and 8-deficient mice.
Interstitial fibrosis reflected as interstitial collagen I expansion
was prominent in both DOCA-treated groups (Figure 3)
. Glomerulosclerosis was marked after
DOCA treatment as assessed by a semiquantitative score of PAS-stained
glomeruli and computer-based analysis of staining for fibronectin and
glomerular collagen IV (Figure 4
; A, B,
and C).
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8-deficient and 129
wild-type mice (not shown). For that reason C57/BL6 and 129xC57/BL6
intercrosses were not chosen for further investigations.
DOCA-Treated 8 Integrin-Deficient Mice Suffer from Extensive
Glomerular Disruption
In the 8-deficient mice, glomerular cells were in a more
activated state compared to wild types, as shown by immunoreactivity
for smooth muscle actin. This was particularly prominent after
treatment with DOCA (Figure 5A)
. There
was a tendency toward formation of microaneurysms (as assessed by a
score for capillary widening) in the salt-loaded 8-deficient mice
and most prominently in DOCA-treated 8-deficient mice, compared to
wild types (Figure 5B)
. The glomerular tuft appeared much more
disrupted in DOCA-treated 8-deficient mice than in wild types where
DOCA treatment did not lead to mesangiolytic lesions (Figure 6, B and E)
. The DOCA salt-treated 8
integrin-deficient group had the highest degree of glomerular fibrin
deposition (7.2 ± 1.1% of glomeruli), indicating widespread
exudation of serum from the glomerular capillaries (Figure 6F)
. Much
less fibrin-positive glomeruli were detected in DOCA salt-treated wild
types (2.4 ± 0.8%) as depicted on Figure 6E
. Salt-loaded control
groups were always negative for fibrin (not shown).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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8 integrin is important for the
maintenance of structural integrity of the glomerulus after mechanical
injury: 1) 8 was more abundant in glomeruli of DOCA salt-treated
mice than in control mice; and 2) 8-deficient mice displayed a
degree of glomerular disruption in this model of glomerular
hypertension that was not found in any other mouse strain investigated.
Cell-matrix interactions are conceivably important in the adaptation of
the vascular wall to high blood pressure. However, surprisingly little
information has been reported on integrins in hypertension. Most of
these studies focused on leukocyte adhesion,12
platelet
glycoproteins,13
and the cellular transduction of
mechanical signals.14
Intengan and
co-workers15
reported that vß3 and 5ß1 integrins
are up-regulated in the vascular wall of a genetic model of
hypertension. Together with our observation that 8ß1 is induced by
DOCA salt hypertension, these data suggest that the induction of
integrin receptors that mediate contact to extracellular matrix
proteins such as fibronectin or vitronectin is part of the adaptive
response of the blood vessel wall to high blood pressure.
Our data suggest that lack of 8ß1 leads to a destruction of
capillary tufts in the presence of high blood pressure. The absence of
8ß1 can apparently not entirely be compensated for by other
integrins. We are not aware of functional studies on the role of other
integrins in hypertension. One recent study reported that endothelin-1
decreased v integrin expression.16
In view of our
results, it is intriguing to speculate that the down-regulation of
integrin expression may contribute to the vasculotoxic effects of
endothelin-1.17
Altered glomerular expression patterns of integrins have been more
extensively described in various forms of glomerulopathies. In IgA
nephropathy, the vitronectin receptor vß318
and the
fibronectin receptor 5ß119
were strongly enhanced in
the mesangium. Similar changes were observed in lupus
nephritis.18,19
During development of diabetic
nephropathy, the glomerular expression of integrins such as 3, 5,
or v is profoundly changed.20
These findings suggest an
important role for integrins in pathological rearrangements of the
matrix and alterations in cell behavior during glomerular diseases.
Whether or not 8 integrin expression also undergoes pathological
changes in human glomerulopathies has not yet been studied. In a
transient rat model of glomerulonephritis, an induction of mesangial
8 integrin after the onset of glomerular injury was found that was
reduced to control levels after complete healing of the
glomerulus.7
The structure of the glomerulus is established and maintained by
interactions of glomerular cells with the adjacent extracellular
matrix. In the mesangial matrix surrounding mesangial cells, several
possible ligands for 8 are present, including fibronectin as the
most abundant and vitronectin as well as tenascin C.21
During DOCA-induced glomerulosclerosis, many matrix constituents are
up-regulated and the 8 ligand osteopontin is expressed de
novo by glomerular cells.11
8 can promote cell
attachment to matrix,22
and ligands for 8 are
up-regulated in glomeruli of hypertensive animals. Presumably, lack of
8 in our model weakens the attachment of mesangial cells to the
mesangial matrix, leading to a loss of tissue integrity in the presence
of mechanical stress. Further, signaling events induced by
matrix-integrin interaction contribute to cell differentiation in many
instances.23,24
Therefore, lack of 8 on mesangial cells
could contribute to the activation of these cells to a fibroblast-like
phenotype (induction of smooth muscle actin) as described by Johnson
and colleagues.25
Interestingly, the increase in fibronectin is comparable in
DOCA-treated wild-type and 8-deficient mice and very similar to the
increase of collagen IV, which is not a ligand for 8. Thus, the most
abundant ligand for 8 in the mesangial matrix is regulated
independently of the presence of its integrin receptor 8.
Transforming growth factor (TGF)-ß is thought to play a central role
in renal fibrotic events.26
This seems also to be true for
the DOCA salt model of hypertension in which expression of TGF-ß is
up-regulated after 6 weeks of disease.27
TGF-ß can
induce expression not only of matrix constituents but also of matrix
receptors in glomerular cells.21,28
Several studies have
shown that expression of 8 is enhanced by TGF-ß in cultured
mesenchymal cells,7,29
including renal mesangial
cells.7
Whether the increase in glomerular
immunoreactivity for 8 in DOCA salt hypertension is because of the
up-regulated TGF-ß expression in this disease model remains to be
shown. Other factors, such as mechanical stress, could possibly also
contribute to the increase in glomerular 8.
Similar to 3-deficient mice, in which capillary loop widening was
described,30
8-deficient mice showed a tendency toward
microaneurysm formation that was clearly enhanced by superimposing
mechanical stress on glomerular capillaries. However, in 3-deficient
mice, this defect seems to be produced by podocyte
failure,30
whereas in 8-deficient mice probably the
integrity of the mesangium is weakened.
To compensate for the lower renal mass, we uninephrectomized wild-type
but not 8-deficient mice. The measurements of relative kidney weight
and creatinine clearance indicate that uninephrectomy in wild types led
to very similar renal mass and function as in 8-deficient mice. We
cannot exclude the possibility that the subtle glomerular alterations
induced by the lack of 8 influence the degree of hypertensive
glomerular damage. However, we consider it unlikely that these
alterations alone can account for the extensive destruction of the
glomerular capillary architecture in 8-deficient DOCA-treated mice.
A recent publication by Levine and colleagues31
investigated 8 integrin expression in the context of fibrotic
mechanisms in fibroblast cells of lung and liver. 8 expression by
lung alveolar interstitial cells was greatly enhanced in fibrosis.
Hepatic stellate cells displayed a de novo expression of
8 when fibrosis was induced by bile duct ligation or carbon
tetrachloride injury. Many of the 8-positive myofibroblast cells
co-expressed smooth muscle actin, which has been implicated in the
development of fibrosis in several organs.32,33
Integrins
could play an important role in the activation of fibroblasts by
signals from the extracellular matrix. In the normal kidney of mouse,
rat, and man 8 was not detected in the tubulointerstitium. However,
in the renal interstitium of some DOCA-treated animals clusters of
fibroblasts became reactive for 8, similar to the reported findings
in liver,31
suggesting a role of 8 also in renal
interstitial fibrosis.
Together, these findings indicate a role for 8 integrin in fibrotic
and/or sclerotic processes in many organs, including liver, lung, and
kidney. Moreover, our data support the notion that 8 may also
contribute to adaptive changes that are necessary in the presence of
mechanical stress.
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Acknowledgements |
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Footnotes |
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Supported by grant SFB 423/A2 from the Deutsche Forschungsgemeinschaft.
Prof. R. Bernd Sterzel died August 6, 2001.
Accepted for publication November 14, 2001.
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, P < 0.05, DOCA treated
