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Regular Articles |
From The First Department of Medicine*
and the
Hemodialysis Unit,
Hamamatsu University School
of Medicine, Hamamatsu, Shizuoka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth
muscle actin-positive myofibroblasts as well as ED 1-positive
monocytes/macrophages were examined. Necrotic tubules initially
appeared around the corticomedullary junction after uranyl acetate
injection, then spread both downstream and upstream of proximal
tubules. Peritubular -smooth muscle actin-positive myofibroblasts
appeared and extended along the denuded tubular basement
membrane, establishing network formation throughout the cortex
and the outer stripe of outer medulla at days 4 to 5. Tubular
regeneration originated in nonlethally injured cells in the distal end
of S3 segments, which was confirmed by lectin and
immunohistochemical staining using markers for tubular segment.
Subsequently, upstream proliferation was noted along the
tubular basement membrane firmly attached by myofibroblasts. During
cellular recovery, no entry of myofibroblasts into the tubular
lumen across the tubular basement membrane was noted and only a few
myofibroblasts showed bromodeoxyuridine positivity. The fractional area
of -smooth muscle actin-positive interstitium reached a peak
level at day 7 in the cortex and outer stripe of outer medulla,
then gradually disappeared by day 15 and remained only around dilated
tubules and in the expanded interstitium at day 21. ED 1-positive
monocytes/macrophages were transiently infiltrated mainly into the
region of injury. They did not show specific association with initially
necrotic tubules, but some of them located in close proximity
to regenerating tubules. Nonlethally injured cells at the distal end of
proximal tubules are likely to be the main source of tubular
regeneration, and the transient appearance of interstitial
myofibroblasts attached to the tubular basement membrane immediately
after tubular necrosis might play a role in promoting cellular recovery
in possible association with monocytes/macrophages in uranyl
acetate-induced acute renal failure.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cellular recovery after acute renal failure (ARF) is a unique form of wound healing or tissue remodeling, and complete regenerative repair can occur. It is possible that interstitial myofibroblasts also participate in the regenerative repair in ARF based on their potential role in wound healing. In this regard, embryonic differentiation of the kidney requires interaction between the mesenchyme and epithelium.4 During nephrogenesis, a part of the renal mesenchyme that responds to a signal from the ureteral epithelium is converted into a new epithelium, kidney tubules.4 The source of regenerating cells in ARF is thought to be tubular epithelial cells at both ends of the necrotic (damaged) segment of the nephron, as well as other isolated surviving cells along the denuded surface of the tubular basement membrane (TBM).5 However, the exact origin of regenerating cells and the sequence of cellular recovery after acute tubular damage are not well known. Therefore, it is important to understand the role of renal interstitial myofibroblasts in the cellular recovery in ARF and the pathway involved in such process.
To investigate the above issues, we used in the present study a rat
model of ARF induced by injection of uranyl acetate (UA). Specifically,
we investigated the serial changes in the distribution of tubular
necrosis and regenerating cells, ie, bromodeoxyuridine
(BrdU)-incorporating cells or vimentin-expressing cells, and their
temporal and spatial relationship to interstitial cells expressing
-smooth muscle actin (-SMA; myofibroblasts) as well as
interstitial monocytes/macrophages. In addition, we determined the
origin of the initial regenerating cells using lectins and
immunohistochemical markers for nephron segments. Our results indicate
that tubular regeneration commences in nonlethally injured cells in the
distal end of S3 segment of the proximal tubule (PT) and that transient
appearance of interstitial myofibroblasts might play a role in cellular
recovery in UA-induced ARF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 50 male Sprague-Dawley rats weighing 230 to 300 g (SLC Co., Shizuoka, Japan) were used in the present study. Rats had free access to standard rat chow and drinking water. They received a single intravenous injection of 5 mg/kg of UA via the dorsal penile vein, and five rats were sacrificed before and at 2, 2.5, 3, 4, 5, 7, 9, 15, and 21 days after UA injection. To label cells that actively synthesize DNA, all rats were injected intraperitoneally with 40 mg/kg BrdU (Sigma Chemical Co., St. Louis, MO) 1 hour before sacrifice.6 Rats were anesthetized with intraperitoneal pentobarbital sodium (30 mg/kg), and a blood sample was collected through the abdominal aorta, then both kidneys were removed after flushing with phosphate-buffered saline (PBS). Serum creatinine level was measured by the enzymatic method (Mizuho Med., Saga, Japan).
Immunohistochemistry and Lectin Histochemistry
The kidneys were bisected through longitudinal axis and were
fixed with either 4% paraformaldehyde or methacarn solution and
embedded in paraffin. For histological examination of renal tissues,
4-µm sections were stained with periodic acid-Schiff (PAS). A direct
or indirect peroxidase or alkaline phosphatase method was used to
detect a variety of antigens and lectins. This included the
analysis of BrdU, -SMA, vimentin, phaseolus vulgaris
erythroagglutinin (PHA-E),7
aquaporin-1
(AQP-1),8
Tamm-Horsfall protein (THP),9
and
ED 1 monocytes/macrophages.
To detect BrdU, 4% paraformaldehyde-fixed sections were deparaffinized and endogenous peroxidase was blocked by treatment with 3% H2O2 for 30 minutes, then the sections were incubated with normal rabbit serum for 20 minutes, followed by incubation with mouse monoclonal antibody against BrdU (Amersham International, Poole, UK) overnight at 37°C. After washing in PBS, sections were incubated with peroxidase-conjugated rabbit anti-mouse IgG (1:50; Chemicon International Inc., Temecula, CA) for 60 minutes at room temperature. The reaction products were visualized by incubation with diaminobenzidine.
To detect PHA-E lectin binding sites, methacarn-fixed sections were deparaffinized and rehydrated then incubated with biotinylated PHA-E lectins (1:800; Sigma) for 60 minutes. After washing in PBS, sections were incubated with Vectastain ABC-AP reagent, an avidin-biotin-alkaline phosphatase complex (Vector Laboratories, Burlingame, CA) for 30 minutes. Finally, the Vector Red Alkaline Phosphatase Substrate Kit I (Vector Laboratories) was used as chromogen in which 1 mmol/L levamisole (Sigma) was added to block endogenous alkaline phosphatase. All incubations were performed at room temperature.
To detect other antigens, a standardized ABC technique was performed by
using the antibodies listed in Table 1
.
Briefly, sections were incubated with the primary antibody and reacted
with the biotinylated secondary antibody for 30 minutes at room
temperature. Then, streptavidin-conjugated peroxidase (Nichirei, Tokyo,
Japan) or Vectastain ABC-AP reagent was added for 30 minutes.
Visualization was performed after BrdU or lectin staining.
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-SMA and BrdU was performed on
the sections at day 5. Sections were first stained for -SMA using
the Vectastain ABC-AP kit with Vector Red, then stained for BrdU using
the diaminobenzidine detection system. Consecutive sections were also
used for comparison of different staining patterns. Counterstaining, if
necessary, was performed by using hematoxylin or methyl green. For control sections, the first antibodies were omitted or replaced by the normal serum of corresponding animal and signals in both control and experimental sections were negative or negligible.
Immunoelectron Microscopy for Detection of -SMA
Cortical tissues fixed with 4% paraformaldehyde were dehydrated
in a graded ethanol series and embedded in Unicryl (British BioCell,
Cardiff, UK) at -30°C according to the instructions provided
by the manufacturer. Ultrathin sections were incubated with mouse
anti-human -SMA (DAKO, Carpinteria, CA) diluted 1:100 in PBS, and
subsequently, with rabbit anti-mouse IgG (Chemicon) diluted 1:500 in
PBS, and goat anti-rabbit IgG conjugated with 10 nm gold (Ultra
Biosols, Liverpool, UK) diluted 1:10 in 0.02 mol/L Tris-HCl buffer (pH
7.0). Sections were stained with UA then examined by a JEM-1220
electron microscope (JEOL, Tokyo, Japan).
Morphometric Analysis of Location of Tubular Necrosis and of BrdU-Positive Tubular Cells
To examine the serial changes in the distribution of PT necrosis,
areas of PAS-stained longitudinal sections of the kidney obtained at
days 2, 2.5, 3, 4, 5, and 7 were projected onto a TV monitor. The
viewed area of each section was 
6.4 mm2
in
each rat. The cross-section of PT with severe damage (representing
desquamation of tubular epithelial cells associated with focal
granulovacuolar epithelial cell degeneration amounting to 75 to 100%
of the section) was counted as necrotic tubules. The location of
targeted PT in the cortex (CO) or the outer stripe of outer medulla
(OSOM) was expressed as A/B (0 to 1.0) using a computer-assisted image
analyzer, where A represented the distance between the center of PT and
the border of CO-OSOM or OSOM, the inner stripe of outer medulla
(ISOM), respectively, and B the thickness of each kidney layer at the
same site. The number of severe necrotic PT in each 20 arbitrarily
divided zones in each kidney layer was displayed in histograms.
The method for locating BrdU-positive cells in tubules at days 2, 2.5,
3, 4, 5, 7, 9, 15, and 21 was similar to that used for PT necrosis
described above. Areas of BrdU-stained longitudinal sections were
displayed onto the monitor. The viewed area of each section was 10.0
mm2
in each rat. BrdU-positive cells in CO, OSOM,
or ISOM were expressed as A/B, where A represents the distance between
a BrdU-positive cell and the border of CO-OSOM, OSOM-ISOM, or ISOM-the
inner medulla, respectively, and B the thickness of each kidney layer
at the same site. The number of BrdU-positive cells in each 20
arbitrarily divided zones in each kidney layer was displayed in
histograms.
Morphometric Analysis of the Number of BrdU-Positive Cells and of ED 1-Positive Interstitial Monocytes/Macrophages
For semiquantitative analysis, the number of BrdU-positive cells in both tubules and the interstitium or the number of ED 1-positive cells in the interstitium in CO, OSOM, or ISOM at days 2, 2.5 (only for BrdU), 3, 4, 5, 7, 9, 15, and 21 were counted in 50 randomly selected fields of each layer in each kidney at x400 magnification. The mean number at each time point was displayed in histograms.
Morphometric Analysis of -SMA/BrdU Double-Positive Interstitial
Cells
The number of -SMA/BrdU double-positive cells in BrdU-positive
cells or in -SMA-positive cells in the interstitium was counted in
50 randomly selected fields of OSOM at day 5 at x400 magnification,
and the mean percentage of -SMA/BrdU double-positive cells was
calculated.
Morphometric Analysis of -SMA-Positive Cells
For semiquantification of -SMA-positive area in CO, OSOM, and
ISOM at days 2, 3, 4, 5, 7, 9, 15, and 21, point counting was performed
using a routine established method.10
A total of 20 fields
at x400 magnification per section were counted in each animal on a
1-cm2
eyepiece graticule with 10 equidistant grid
lines. Each field was 0.0625 mm2, and the total
area counted per slide was 1.25 mm2. The
percentage of fractional area (percentage of positive area per total
area counted of the section) was calculated using the following
formula: Percentage of fractional area equals number of grid
intersections with positive staining/total number of grid intersections
multiplied by 100.
Statistical Analysis
Data are expressed as the mean ± SEM. Differences between data sets were examined for statistical significance using one-way analysis of variance followed by Fisher’s t-test. A P level <0.05 was accepted as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Scr increased significantly as early as day 3 after UA injection
(P < 0.05 versus level before
induction of ARF), and reached a peak value at day 7
(P < 0.001 versus level before
induction of ARF), then returned to normal level by day 15 (Figure 1)
.
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Although slight morphological changes could be seen throughout the
PT in PAS-stained tissues, the initial lesions of severe necrosis were
predominantly located around the corticomedullary junction where the
denuded TBM was evident as early as day 2 (Figure 2, B and G)
. PT damage with severe
necrosis spread progressively to both downstream and upstream of PT
(Figures 2 and 3)
. Between days 4 and 5,
tubular necrosis was almost maximally distributed in OSOM and CO
(Figure 3)
with cellular debris in the tubular lumen (Figure 2D)
. By
day 7, the majority of necrotic tubules disappeared and denuded TBM
were almost covered with regenerating cells with flattened cytoplasm
(Figure 2, E and H)
, but there were still few clusters of severely
damaged tubules (Figure 2E)
. After day 7, stratification of
hyperplastic PT with focal papillary projections could be seen in OSOM
(Figure 2, E
and I). By day 21, desquamation of epithelial cells in
papillary clusters was noted in the tubular lumen, and
hyperproliferation was almost remodeled with occasional tubular
dilation (Figure 2F)
.
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Location and Kinetics of BrdU-Positive Cells in Tubules
The majority of BrdU-positive regenerating cells initially
appeared at the border zone between OSOM and ISOM as early as day 2
(Figures 4B and 5)
. However, there was a sudden sharp
rise in the number of these cells at day 2.5 (Figure 5)
. To identify
the origin of BrdU-positive cells, double-staining of BrdU and AQP-1 or
BrdU and THP was performed in consecutive sections at day 2. Although
AQP-1 is positive in the apical brush border and in basolateral
membrane of PT, as well as in both apical and basolateral membrane of
TDL in the normal kidney,8
heavy immunostaining is
considered to be a marker for TDL. The majority of BrdU-positive cells
around the border between OSOM and ISOM was almost confined to tubular
epithelial cells, but not to interstitial cells. These BrdU-positive
cells were not located in heavy AQP-1-positive staining tubules (Figure 6A)
or in THP-positive staining tubules
(marker for TAL) (Figure 6B)
. In addition, most BrdU-positive cells
showed minor morphological change (Figure 6C)
in the PTs with weak
AQP-1 positivity (Figure 6A)
. However, a small number of BrdU-positive
cells was found in both AQP-1- and THP-negative staining cells (Figure 6B)
, indicating collecting duct cells which was also judged by
morphology. It is unlikely that BrdU-positive collecting duct cells are
the source of initial regenerating cells, which will proliferate to
cover the PT damage, because collecting ducts are not connected with PT
segments directly. These findings suggested that the majority of the
initial regenerating cells originated from nonlethally injured cells at
the distal end of S3 segments of PT. A small number of TAL also showed
BrdU-positivity, especially in the late phase of this model (data not
shown). Morphometric analysis revealed that the frequency distribution
of BrdU-positive tubular cells was shifted from the inner to the outer
zone of OSOM by day 5 (Figures 4 and 5)
. These findings suggest the
upstream proliferation of regenerating cells from the end of the S3
segment of PT. The BrdU activity in OSOM subsided at day 7 (Figures 4F and 5)
when the regenerating cells almost covered the denuded TBM as
noted in PAS-stained sections (Figure 2H)
. Parallel to the
hyperproliferation in OSOM (Figure 2I)
, the second peak of
BrdU-positive cells appeared at day 9 (Figure 4G and 5)
, but decreased
by day 21 (Figure 5)
. Determination of the mean number of BrdU-positive
cells per field also showed a biphasic BrdU-activity in tubular cells
in OSOM (Figure 7)
.
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. A small number of
distal tubules also showed BrdU-positivity in CO (data not shown). In
ISOM, a significant number of BrdU-positive cells was noted as early as
day 3, but the total number of such cells was less than that in OSOM
(Figures 5 and 7)
. The majority of these cells were TAL and collecting
duct cells, which were also judged by morphology. The frequency
distribution of BrdU-positive cells seemed to be shifted from the outer
to the inner zones of ISOM (Figure 5)
and BrdU activity in ISOM showed
a biphasic appearance, similar to that in OSOM (Figures 5 and 7)
. Vimentin Expression in Tubules
We used vimentin expression as a marker for tubular regeneration
and/or dedifferentiation as reported previously.11
In
normal kidney, vimentin was expressed in glomerular epithelial cells,
mesangial cells, and vascular endothelial cells and to a lesser extent
in interstitial cells, but tubular epithelial cells did not express
vimentin (Figure 8A)
.11,12
Vimentin-positive tubular cells in OSOM appeared as early as day 2
exclusively in the inner zone of the OSOM (Figure 8B)
. High-power
magnification showed these to be spindle-like large cells containing
oval-shaped nuclei (Figure 8H)
, extending mainly upstream of the
nephron segments in OSOM (Figure 8)
, similar to PT regeneration
observed by BrdU (Figures 4 and 5)
. Vimentin was also positive in
initial BrdU-positive regenerating PT (Figure 6C)
. These findings
confirmed upstream proliferation of the S3 segment of PT during the
recovery phase at day 7 when most TBM were covered with flattened PT
cells showing intense vimentin staining (Figure 8F)
. Intense vimentin
staining could be found in PT until day 9 (Figure 8G)
, then gradually
disappeared. Hyperproliferative PT also expressed vimentin (Figure 8, F, G, and J)
after day 7. When PHA-E, a constituent molecule of brush
border,7
was used as a marker of differentiated apical
membrane of PT, totally negative staining for PHA-E was found in
regenerating PT until day 5, but slight staining for PHA-E reappeared
in PT at day 7 (Figure 9)
. These findings
indicated that early-redifferentiated PT cells after proliferation do
not fully recover at least until day 9.
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BrdU-Positive Cells in Interstitium
The number of BrdU-positive cells in the interstitium in CO, OSOM,
and ISOM also increased significantly as early as days 5, 4, and 4,
respectively, and showed biphasic peak at days 5 and 9 (Figure 7)
. The
peak points of BrdU activity in the interstitium were similar to that
in the tubules (Figure 7)
, but the number of BrdU-positive cells in the
interstitium was less than in tubules, the second peak was higher than
the first one, and significant appearance of these cells in the
interstitium of CO, OSOM, and ISOM did not precede that in tubules
(Figure 7)
. Moreover, the distribution of BrdU-positive cells in the
interstitium in OSOM did not show a regular pattern and temporal
association with regenerating tubules.
Appearance of Interstitial Myofibroblasts
In normal rats, -SMA protein was detected in vascular smooth
muscle cells but not in glomeruli, tubules, or interstitial cells
(Figure 10A)
. -SMA-positive
interstitial cells, indicating myofibroblasts, were visible in OSOM as
early as day 2 (Figure 10B)
. Initially they appeared exclusively around
damaged tubules with denuded TBM which were located mainly in the outer
zone of the OSOM (Figure 10B)
and extended along with the progression
of severe tubular damage (Figures 2 and 10)
. Between days 4 and 5,
myofibroblasts formed a network around necrotic tubules throughout the
cortex and medulla as if they structurally supported the frame of the
denuded TBM (Figures 10D and 11A)
. The
exact location of myofibroblasts when they entered the tubular lumen
was not clear under light microscopic examination (Figure 11A)
.
Immunoelectron microscopy revealed that the cell processes labeled with
gold particles, indicating -SMA, were firmly attached to the outer
surface of the denuded TBM and formed a network appearance by day 5
(Figure 12)
. Entry of myofibroblasts
into tubular lumen across TBM was not evident at the ultrastructural
level (Figure 12)
. We performed -SMA/BrdU double-immunostaining in
OSOM at day 5 (Figure 13)
when
interstitial myofibroblasts are developing network formation. Among
BrdU-positive interstitial cells, only 2.5 ± 0.5% showed
-SMA-positivity, and among -SMA-positive cells, only 1.5 ±
0.3% expressed BrdU positivity, suggesting that most BrdU-positive
interstitial cells were not myofibroblasts and that myofibroblasts
seemed not to undergo cell proliferation actively. Most peritubular
-SMA expression was transient in OSOM and CO and began to decrease
from day 9 (Figure 10F)
, became faint by day 15 (Figure 10H)
, and
almost negative by day 21 (Figure 10I)
. However, -SMA staining in
focally expanded interstitium, around hyperplastic tubules (Figure 10G)
and dilated tubules (Figure 10I)
, was seen at day 7 and thereafter. By
day 21, -SMA-positive cells were present only around dilated tubules
(Figure 10I)
and in small areas of focally expanded interstitium
(Figure 10I)
. Periglomerular -SMA staining was noted at day 7 and
became most prominent at day 15 (Figure 10H)
but diminished rapidly by
day 21. -SMA-positive staining in the expanded interstitial area and
periglomerular area was confirmed by increased fractional areas of
-SMA in the later phase of this model
(Figure 14)
.
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-SMA extended from the OSOM,
and significantly increased in the outer zone of ISOM at day 5, then
extended to the whole ISOM, reaching a peak level at day 9 (Figure 14)
.
In normal rats, only a faint vimentin staining could be seen in some
renal interstitial cells in OSOM. After induction of ARF, the number of
faint vimentin-positive peritubular interstitial cells increased
throughout OSOM as early as day 2 (Figure 8I)
. This was followed by
further increases in intensity of staining (Figure 11B)
. After day 4,
the serial changes in vimentin-staining in the interstitium were
similar to those of -SMA staining (Figure 11, A and B)
. These
findings suggest that interstitial cells may firstly express vimentin
then acquire -SMA.
Interstitial ED 1-Positive Monocytes/Macrophages
ED 1-positive monocytes/macrophages were found only occasionally
in control kidneys. With the development of PT damage after induction
of ARF, interstitial ED 1-positive cells increased in the region of
injury, then gradually disappeared after tubules have regenerated (data
not shown). Specific accumulation of ED 1-positive cells closely around
the initially necrotic PT with the denuded TBM was not evident, and the
distribution of ED 1-positive cells did not show a regular pattern in
CO, OSOM, and ISOM. However, some ED 1-positive cells located in close
proximity to myofibroblasts surrounding regenerating tubules (Figure 11C)
after day 3. Morphometric analysis revealed that interstitial ED
1-positive cells in CO, OSOM, and ISOM significantly increased as early
as day 3, peaking at days 5, 4 to 7, and 5, respectively
(Figure 15)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is well known that PTs are frankly damaged in ARF induced by nephrotoxins such as gentamicin,13 mercury,14 cisplatinum,15 and organic compounds like petroleum hydrocarbons.16 In general, the S3 segment of PT is more severely damaged than the S1 and S2 segments in the majority of nephrotoxin-induced ARF with the exception of gentamicin and petroleum hydrocarbons.13,16 In our model of UA-induced ARF, PT necrosis initially occurred in the initial part of the S3 segment in the outer OSOM and slight morphological changes were already present throughout the whole S3 segment as early as day 2. Necrotic tubules progressively spread from the corticomedullary junction toward both downstream and upstream of PT, and were maximally distributed almost throughout the whole S3 segment by days 4 to 5. To our knowledge, detailed description of the progression of PT necrosis has not been previously described in nephrotoxin- and ischemia-induced ARF.
In the present study, we provided additional evidence regarding the origin of regenerating cells and the mode of cellular recovery. The initial regenerating tubular cells (BrdU- or vimentin-positive cells) were found at the distal end of the S3 segment as early as day 2. This was confirmed by using markers for nephron segments. The first group of regenerating cells showed partial cellular damage. Subsequently, regenerating cells proliferated toward upstream of proximal nephron in OSOM and almost covered the denuded TBM by day 7. Based on mesenchymal-epithelial cell transdifferentiation during nephrogenesis,4 it is possible that interstitial multipotent stem cells could trans-differentiate into tubular epithelial cells under pathological conditions. In this study, however, during cellular recovery, electron microscopic examination revealed the lack of invasion of myofibroblasts across the TBM into tubular lumen around the distal ends of S3 segments and even throughout the proximal nephron in OSOM. These findings suggest that the nonlethally injured PT cells themselves at the distal end of the S3 segment may be the main source of cellular recovery. In most nephrotoxin- and ischemia-induced ARF, cellular recovery was reported to originate from surviving cells in the necrotic zone or viable cells adjacent to the necrotic zone.4,13,17 It is conceivable that isolated surviving cells are responsible for cellular recovery after tubular damage when the degree of the latter was low and many isolated surviving cells were still present in these areas. This seems not to be the case in our model. Only Haagsma et al18 reported that regeneration commenced at both ends of damaged PT in HgCl2-induced ARF. However, they did not elaborate on the mode of progression of cellular recovery. In the present model, BrdU-positive tubular cells in CO appeared later and in a sporadic and irregular pattern than in OSOM, suggesting that the isolated surviving cells mainly participate in cellular recovery in CO. However, it is difficult to assess this issue because the S1 + S2 segments of PT do not run straightly from the junction of the S3 to S2 segment to the glomerulus.19 It remains unclear at this stage whether the cells at the distal end of PT are characteristically resistant to UA-insult or are proliferatively potent. Furthermore, the exact stimulus that initiates the proliferative response remains to be determined.
A particularly noteworthy finding in the present study was that the
myofibroblasts appeared and surrounded the initial necrotic PT,
developed along with the progression of PT necrosis, attaching to the
TBM, and formed network throughout the CO and OSOM before acceleration
of cellular recovery. The origin of -SMA-expressing interstitial
cells was not clear, but their immediate appearance adjacent to TBM
strongly suggests that resident stromal cells located adjacent to the
TBM, which initially do not express features of smooth muscle cell
differentiation (identified by -SMA), later differentiated to
-SMA expressing cells. The mechanisms responsible for such
phenotypic modulations are unknown, but recent studies suggest that
transforming growth factor-ß, platelet-derived growth factor, and
extracellular matrix could transform fibroblasts to
myofibroblasts.20
In the present model, injured tubular
cells might secrete cytokines such as platelet-derived growth factor
and transforming growth factor-ß,21,22
resulting in the
activation and proliferation of myofibroblasts. However, in the present
study, analysis of -SMA/BrdU double-immunostaining revealed that
most myofibroblasts did not undergo cell proliferation actively.
Interstitial infiltrating cells are other possible candidates for
cytokine production. Diamond et al23
reported that renal
cortical transforming growth factor-ß1, derived from infiltrating
macrophages, in part, contribute to foster the modulation of
fibroblasts to myofibroblasts within the renal cortex after ureteral
obstruction in rats. In fact, several studies have reported the
presence of infiltrating cells in the interstitium after acute renal
injury.24
In our model, ED 1-positive
monocytes/macrophages did infiltrate transiently in the interstitium.
Specific association of them with initially necrotic tubules surrounded
by myofibroblasts, however, was not evident. In addition, alterations
in components of TBM similar to the extracellular matrix were reported
in ARF.25,26
Moreover, back-leak of filtrate through the
denuded TBM27
and/or relatively high stretching force
acting on the interstitium through the TBM because of loss of tubular
cells and/or tubular obstruction by cell debris, might activate
interstitial cells to express -SMA.
Interestingly, Nagle et al28
observed that interstitial
fibroblasts underwent morphological transformation to smooth
muscle-like interstitial cells in a rabbit model of complete ureteral
obstruction and demonstrated that under these conditions the kidney may
demonstrate increased whole-organ contractility.29
In the
present study, immunoelectron microscopic examination revealed that
myofibroblasts with possible stress fibers closely covered the external
side of TBM as if they provided contractility and resisted the
stretching force. Myofibroblasts have been described in clinical
disorders related to injury and repair phenomena, associated with
prominent tissue retraction and remodeling.20
During wound
healing, fibroblastic cells modulate to a phenotype characterized by
ultrastructural (stress fibers) and biochemical (-SMA) features
typical of smooth muscle cells, indicating that this change is
instrumental in activities such as wound contraction.20
This might also be the case for myofibroblasts during cellular recovery
in ARF. Interstitial accumulation of myofibroblasts is one of the best
predictive indicators of renal fibrosis in progressive renal
disease.1-3
In the present acute tubular injury model,
the majority of peritubular myofibroblasts gradually disappeared after
regeneration of PT, suggesting that they might revert to a quiescent
form or undergo apoptosis.30,31
Although no direct evidence for a role of interstitial cells in ARF is provided in the present study, we postulate that peritubular myofibroblasts play a mediating role in cellular recovery in our model because the regenerating PT underwent proliferation toward upstream of PT along the denuded TBM firmly attached by myofibroblasts. In addition to the possible function of supporting the TBM, myofibroblasts might furnish extracellular matrix components to condition PT proliferation, because fibroblasts play an essential role in the synthesis and regulation of extracellular matrix components in wound healing.20 Moreover, myofibroblasts can provide cytokines as paracrine mitogens, which might promote cellular recovery from ARF. At present, fibroblast growth factors-1 and -7 and hepatocyte growth factor may be possible candidates for cytokines secreted by myofibroblasts in renal interstitium.32-35 Transient interstitial infiltrating cells24,32,36 as well as tubular cells21,22,37-39 are also a possible source of cytokines, which can promote regeneration of the damaged tubules. Indeed, in the present study transient infiltration of interstitial monocytes/macrophages could be found in some association with regenerating tubules.
Regenerative repair of BrdU-positive PT was almost completed by day 7. After day 7, hyperproliferation of PT became prominent and was followed by remodeling of hyperplastic tubules with a resolution of hypercellularity probably through apoptosis, as reported previously.6,40 This proliferation pattern seemed to be reflected by the findings that the biphasic waves of BrdU activities in OSOM reached peak values at days 5 and 9. However, BrdU-positive staining could be also seen in cells stained positive for THP, a marker for the thick ascending limb, especially in the late phase of this model (data not shown). Moreover, although no visible signs of tubular necrosis could be seen in ISOM, a significant number of BrdU-positive cells were also seen as early as day 3. Thus, BrdU-positive cells probably include loop cells and/or collecting duct cells. Although PT is the main site of renal damage in ARF, recent studies indicate that the distal nephron may also react extensively to ischemic41,42 and nephrotoxic13,33 insults, suggesting that the distal nephron might function as part of an adaptive response to the loss of PT function. The significance of BrdU-positive cells in the distal nephron remains to be elucidated. On the other hand, the early changes in vimentin staining in tubules corresponded to those of BrdU, but vimentin stained-cells were noted throughout a long period in PT tubules with or without hyperproliferation at least until day 9, and at day 21, vimentin was still positive in dilated tubules. These findings indicate that vimentin would be a marker for tubular dedifferentiation or injury rather than for regeneration itself as reported previously.11 Although regenerated tubular cells at day 7 showed a weakly positive staining for PHA-E, a constituent molecule of the brush border and a marker of differentiated apical PT membrane, the majority of PT did not show the final differentiation at least until day 9 with respect to vimentin expression.
In summary, we have demonstrated in the present study that nonlethally injured cells at the distal end of S3 segments of PT are likely to be responsible for the cellular recovery in UA-induced ARF. Based on the serial changes in the distribution of tubular damage and peritubular myofibroblasts, as well as their relationship to cellular recovery, we propose that the transient appearance of peritubular myofibroblasts immediately after PT necrosis plays an important role in promoting the regenerative repair.
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Accepted for publication July 1, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Lab Invest 1990, 63:21-29[Medline]
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