close

Вход

Забыли?

вход по аккаунту

?

Podocyte Loss in Aging OVE26 Diabetic Mice.

код для вставкиСкачать
THE ANATOMICAL RECORD 291:114–121 (2007)
Podocyte Loss in Aging OVE26
Diabetic Mice
JENNIFER M. TEIKEN,1 JANICE L. AUDETTEY,1 DONNA I. LATURNUS,1
SHIRONG ZHENG,2 PAUL N. EPSTEIN,2 AND EDWARD C. CARLSON1*
1
Department of Anatomy and Cell Biology, University of North Dakota, Grand Forks,
North Dakota
2
Department of Pediatrics, University of Louisville, Louisville, Kentucky
ABSTRACT
Recent studies show that podocyte nuclear density (NV) and numbers
of renal podocytes per glomerulus (N) are altered in experimental and
spontaneous diabetes mellitus. NV and N are generally reduced, and it
has been hypothesized that these morphological changes may relate to
the loss of glomerular permselectivity in diabetic nephropathy (DN). In
the current study, OVE26 transgenic diabetic mice and age-matched
(FVB) controls (60, 150, or 450 days) were fixed by vascular perfusion
and renal cortical tissues were prepared for morphometric analyses.
ImageJ software and point counting analyses were carried out on light
and transmission electron micrographs to determine glomerular volume
(VG), NV, and N. As expected, mean VG in OVE26 mice increased substantially (134%) over the course of the study and was significantly
increased over FVB mice at all ages. At 60 days, NV and N were not statistically distinguishable in OVE26 and control mice, while at 150 days,
NV was significantly reduced in diabetics but not N. In 450-day-old
OVE26 animals, however, NV and N were both significantly decreased
(231% and 99%, respectively) relative to age-matched FVB mice.
These data suggest that in the OVE26 model of diabetes, significant podocyte loss occurs relatively late in the course of the disease. Moreover, it
seems possible that these podocytic changes could play a role in sustaining the increased permeability of the blood–urine barrier in the later
stages of diabetic renal decompensation. Anat Rec, 291:114–121,
2007. Ó 2007 Wiley-Liss, Inc.
Key words: podocytes; diabetic mice; electron microscopy;
morphometry
Podocytes are structurally important components of
the blood–urine barrier, and recent studies have shown
that they may be critical elements in renal glomerular
permeability (see Shankland, 2006, for review). It has
long been appreciated that podocytes function in the
ultrafiltration of blood to form the renal glomerular filtrate, and they are also known to synthesize components
of the glomerular basement membrane (GBM). In addition, they provide structural stability to the glomerular
tuft (Vogelmann et al., 2003). Although the GBM is
believed to be the main filter in renal permselectivity,
podocytic slit diaphragms perform the final ultrafiltration step and collectively form a size-selective barrier
maintained by several proteins including nephrin, podocin, CD2AP, neph-1, a-actinin4 (Asanuma and Mundel,
2003) FAT, P-cadherin (Paventstädt et al., 2003), and
Ó 2007 WILEY-LISS, INC.
Grant sponsor: North Dakota Lions Foundation; Grant sponsor: PHS; Grant number: DK072032; Grant sponsor: JDRF;
Grant numbers: 1-2005-88 and 3-2005-932.
*Correspondence to: Edward C. Carlson, Department of Anatomy and Cell Biology, University of North Dakota, School of
Medicine and Health Sciences, Room 1701, 501 North Columbia
Road Stop 9037, Grand Forks, ND 58202-9037.
Fax: 701-777-2477. E-mail: ecarlson@medicine.nodak.edu
Received 6 September 2007; Accepted 2 October 2007
DOI 10.1002/ar.20625
Published online in Wiley InterScience (www.interscience.wiley.
com).
PODOCYTE MORPHOMETRY IN DIABETIC MICE
ZO-1 (Rincon-Choles et al., 2006). Slit diaphragms and
their components are vital for the maintenance of
healthy podocytes and also for regulating the passage of
protein (Marshall, 2005). Disruptions in the filtration
process by diseases such as diabetes may lead to severe
proteinuria and/or the passage of other plasma proteins
normally retained by the podocytic slit diaphragm.
Morphological hallmarks of diabetic nephropathy (DN)
include GBM thickening (Adler, 1994; Viberti et al.,
1994; Goode et al., 1995), expansion of the mesangial
region (Steffes et al., 1989), and an accumulation of mesangial matrix (Osterby et al., 2001). More recently, however, alterations in podocyte number, structure, and
function have been shown to occur with the onset and
progression of diabetes, and this cell type has recently
received increased attention (Pagtalunan et al., 1997;
Kriz et al., 1998; Mifsud et al., 2001; Shirato et al.,
2001; White et al., 2002; Dalla Vestra et al., 2003; Vogelmann et al., 2003; Petermann et al., 2004; Marshall,
2005; Wolf et al., 2005; Yu et al., 2005; Shankland, 2006;
Susztak et al., 2006; Macconi et al., 2006). Podocytes
typically are reduced in number in diabetes, and in
patients some podocytes are recoverable in the urine as
viable cells (Vogelmann et al., 2003).
The OVE26 transgenic diabetic mouse model was originally created by Epstein and coworkers (1989, 1992)
and displays many of the characteristics of human diabetes. These characteristics include significant nephropathic features, which have been well characterized. As
examples, OVE26 kidney weights were almost doubled
between 2 and 5 months (Zheng et al., 2004), and morphometric analyses have shown an increase in GBM
thickness (Carlson et al., 1997; 2003), glomerular volume, and mesangial matrix area (Zheng et al., 2004).
Interestingly, significant albuminuria was frequently
seen by 2 months of age, and urinary albumin excretion
(UAE) rates increased progressively with age (Zheng
et al., 2004).
Despite these compelling changes in renal morphology
and physiology, podocyte morphometry has not been
investigated in the OVE26 diabetic model. Accordingly,
we hypothesized that podocyte density and number are
reduced progressively in these mice. In the current
study, morphometric analyses were carried out on light
(LM) and transmission electron microscopic (TEM)
images to determine mean glomerular volume (VG) and
mean podocyte nuclear density (NV), from which average
number of podocytes per glomerulus (N) was calculated.
The results of our study show for the first time significant podocyte loss in OVE26 mice relative to agematched controls.
MATERIALS AND METHODS
Experimental Animals
Control (FVB) and transgenic diabetic (OVE26) mice
or fixed renal tissues were received from the laboratories
of Dr. Paul Epstein, Department of Pharmacology and
Toxicology, University of North Dakota, Grand Forks,
North Dakota, and Department of Pediatrics, University
of Louisville, Louisville, Kentucky. Diabetic OVE26 mice
on the FVB background have been maintained for 15
years in Dr. Epstein’s laboratory (Epstein et al., 1992).
All animals had free access to Harlan Teklab Rodent
Diet #8640 and tap water, and no insulin therapy was
115
given. All animal procedures adhered to the guidelines
of the NIH Guide for the Care and Use of Laboratory
Animals and were approved by the IACUC committees
of the University of North Dakota and the University of
Louisville.
Tissue Preparation and Microscopic Technique
Tissues from at least three OVE26 animals (60, 150,
and 450 days of age), and at least three age-matched
FVB mice (20 total animals) were used in this study.
Before killing, all mice were weighed, and blood glucose
levels were determined by ‘‘Lifescan’’ glucometer. All
animals were killed by vascular perfusion with cold Karnovsky’s (1965) fixative following sodium pentobarbital
anesthesia. Kidneys from all animals were removed,
weighed, and decapsulated. They were sliced longitudinally and the medulla from each slice was removed and
discarded. The remaining cortical strips were cut into 1mm3 tissue blocks, and 10–15 blocks from each kidney
were selected randomly by unbiased technical personnel
and placed in cold fixative for at least 2 hr. This process
was followed by post-fixation in OsO4, dehydration, and
embedding in Epon/Araldite. Thick (250 nm) sections
were made and stained with toluidine blue for LM observation, and thin (silver–gray interference color) sections
were mounted on Formvar-coated copper slot grids and
stained with uranyl acetate and lead citrate for conventional TEM. All TEM specimens were observed and photographed in a Hitachi 7500 TEM. For TEM calibration,
a standard cross-grating (2,160 lines/inch) was photographed at the same optical conditions as were the tissues. All TEM prints were made at a constant enlarger
setting to produce identical final magnifications of 1,960.
Podocyte Morphometry
Mean glomerular volume (VG). All data were
derived from LM micrographs of renal cortical thick sections taken at 5-mm intervals. At least 15 and up to 20
glomerular profiles from each animal age and type (720
total LM images) showing well-perfused and patent glomeruli were chosen randomly by unbiased observers.
Glomerular profiles were digitally imaged from each tissue block, and mean glomerular profile areas for each
animal were determined using ImageJ software. VG was
calculated by the method of Weibel and Gomez (Weibel
and Gomez, 1962; Weibel, 1979) from the formula: VG 5
Area1.5 3 b /K, or VG 5 Area1.5 3 1.38/1.01, (equation 1)
where b (shape coefficient for a sphere) is 1.38, and K
(size distribution coefficient assuming a 10% coefficient
of variation) is 1.01.
Mean podocyte nuclear density (NV). All data
were derived from TEM images of glomerular profiles.
Assuming one nucleus per cell, podocyte nuclear density
(i.e., the number of podocyte nuclei per unit of glomerular tissue, NV) and podocyte density are the same.
Therefore, determining NV in TEM micrographs provides the desired numerical data and also conveniently
obviates the difficulty of quantifying variable and complex podocytic cell processes, which may belong to the
same cell. Also, unequivocal identification of podocyte
nuclei is facilitated at the level of ultrastructure.
In an effort to avoid bias produced by variable nuclear
shape and size distribution, the Weibel and Gomez
116
TEIKEN ET AL.
Fig. 1. A,B: Low-power transmission electron micrographs (TEMs) of glomerular profiles in FVB (A)
and OVE26 (B) mice at 450 days of age. TEM resolution is sufficient for clear recognition of podocyte
nuclei (arrows) in both genotypes. 3800.
(Weibel and Gomez, 1962; Weibel, 1979) method as
described by White and Bilous (2004) was used in the
current study. By this technique, point counting measurements were carried out on TEM micrographs of randomly chosen sections of entire glomerular profiles similar to those shown in Figure 1. Measurements were performed on 9–14 TEM sectional profiles from animals of
each age and type (65 total TEM images). Podocyte NV
was determined using the formula: NV 5 K/bH (NA3/VV)
(equation 2). A transparent plastic grid with fine points
11.5 mm apart was superimposed on each TEM glomerular profile, and the number of podocyte nuclear profiles
(n) and fine points hitting the glomerular tuft (Ptuft)
were counted to estimate podocyte nuclear profile area
(NA): NA 5 Sn / [S Ptuft 3 area per point] (equation 3).
Fine points hitting the glomerular tuft at all ages averaged 125.3 in FVB mice and 196.7 in OVE26 animals.
The number of fine points hitting podocyte nuclear profiles (Pnucleus) were then counted to calculate the volume
fraction of podocyte nuclei in the glomerular tuft (VV):
VV 5 SPnucleus /SPtuft (equation 4). Fine points hitting
podocyte nuclei at all ages averaged 4.3 in both FVB
and OVE26 mice. Podocyte NV was then calculated from
equation 2, with a size distribution (K) of 1.01 and a
shape constant (b) of 1.55.
Mean podocyte number per glomerulus (N).
For determination of N, we used the general formula for
numerical density: N 5 VG 3 NV, using VG from equation 1 and NV from equation 2.
used. Statistical analyses were carried out using SigmaStat 3.0 (SPSS, Inc.).
RESULTS
Blood Glucose
Blood glucose levels in nonfasted FVB control animals
at all ages ranged from 84–230 mg/dl and averaged
134.5 mg/dl. As early as 50 days of age, blood glucose
values in OVE26 mice were more than twice that of controls, and by 150 days, OVE26 values were approximately 435 mg/dl. Frequent testing of older diabetic animals confirmed that blood glucose levels remained high
throughout their lifespan (average 443.5 mg/dl) and
occasionally exceeded 500 mg/dl. At each age, blood glucose was significantly higher in OVE26 mice than in
age-matched controls.
Body Weight
Between 60 and 150 days average body weights of
FVB mice increased from 23.8 g to 32.7 g (Table 1).
However, by 450 days, their weights decreased slightly
to 30.9 g (total weight gain 30%). Similarly, body
weights in the OVE26 model increased between 60 and
150 days from 21.7 g to 29.5 g, and then decreased at
450 days to 28.5 g (total weight gain 31%). Mean
OVE26 body weights were slightly lower than FVB body
weights in all age groups.
Kidney Weight
Statistics
Data were analyzed using the unpaired t-test. Level of
significance for all tests was set at P < 0.05 and power
was set at 0.8. In those cases where data did not meet
assumptions of normal distribution and equal variance,
the nonparametric Mann-Whitney rank-sum test was
Mean kidney weights of FVB mice increased 24%
between 60 and 150 days of age (Table 1). Substantial
gains also occurred between 150 and 450 days. In
OVE26 mice, kidney weight increases were even more
dramatic (135%) between 60 days and 150 days, and
the increase was maintained at 450 days. Although
117
PODOCYTE MORPHOMETRY IN DIABETIC MICE
TABLE 1. Mean body and kidney weights of FVB and OVE26 mice 6 SEMa
Mean body weight (g)
Age (days)
60
150
450
Mean kidney weight (g)
FVB
OVE26
FVB
OVE26
23.8 6 1.4
32.7 6 0.5
30.9 6 1.3
21.7 6 1.2
29.5 6 1.1
28.5 6 1.1
0.21 6 0.01
0.26 6 0.01
0.33 6 0.01
0.20 6 0.01
0.47 6 0.02*
0.46 6 0.01*
a
Asterisks indicate that comparisons between FVB and OVE26 show statistical significance
(P < 0.05).
Fig. 2. A,B: Transmission electron micrographs of podocytes (P) in 450-day-old FVB (A) and OVE26
(B) mice. 36,800.
OVE26 mean kidney weights were somewhat lower
(5.0%) than controls at 60 days of age, at 150 and 450
days, mean kidney weights in the diabetic mice were
81% and 39% greater, respectively, than age-matched
normal mice.
Renal Glomerular Morphology
At 60 days of age, renal glomeruli from FVB and
OVE26 mice were indistinguishable by LM and TEM.
However, by 150 days, they were easily differentiated.
At that time, glomeruli in OVE26 mice appeared larger
in diameter than those of FVB controls. Diabetic animals also exhibited thicker GBMs and expanded mesangial regions owing primarily to increased mesangial
matrix. By 450 days, the morphological differences were
further exaggerated (Fig. 1). In FVB mice, podocytes
exhibited typical ultrastructural features at all ages
(Fig. 2A). Primary processes extended from cell bodies
and secondary processes with associated foot processes
showed features considered typical for this cell type.
Although at 60 days podocytes in OVE26 and control
mice were virtually indistinguishable, by 150 days occasional foot process broadening, or effacement was noted
in the diabetics and at 450 days significant effacement
was common in the diabetic mice. In the 450-day-old
diabetics, slits between foot processes were much farther
apart and the narrow cross-sectional foot process profiles
seen in controls were replaced by club-like extensions
that covered large areas of the GBM (Fig. 2B).
Podocyte Morphometry
Mean glomerular volume (VG). Mean glomerular profile areas were determined in mm2 from digital
images using ImageJ software as described in the Materials and Methods section. These were used to calculate
mean VG for FVB and OVE26 mice.
In FVB mice, VG increased slightly (9%) between 60
and 150 days of age (Table 2; Fig. 3A). However, the
increase was much greater between 150 and 450 days
(41%) resulting in a 53% overall increase between
ages 60 and 450 days. As expected, however, changes
were much greater in aging diabetic mice where substantial increases in VG occurred between 60 days and
150 days (111%) followed by a slight rise (11%)
between 150 and 450 days. Between 60 and 450 days,
mean VG increased 134% in the OVE26 strain.
At 60 days, VG differences between OVE26 and FVB
mice were small but significant (19%; P < 0.001). Significant differences occurred also at; both 150 and 450
days (131%; P 5 0.001, and 81%, P 5 0.019, respectively) when VG increased dramatically in the diabetics
(Table 2; Fig. 3A).
Mean podocyte nuclear density (NV). In control FVB mice, mean NV increased slightly between 60
and 150 days (7.29 3 1024/mm3 vs. 7.84 3 1024/mm3),
but then decreased to 5.19 3 1024/mm3 at 450 days,
resulting in an overall decrease in NV with age (Table 2;
Fig. 3B). In OVE26 mice, a substantial mean NV
decrease (86%) occurred from 5.92 3 1024/mm3 at 60
days, to 3.19 3 1024/mm3 at 150 days and further
decreased to 1.57 3 1024/mm3 by 450 days of age (27%
of 60-day value).
At 60 days, mean podocyte NV in OVE26 mice was
slightly less (23%) than in FVB mice. However, at 150
days NV was significantly less (146%; P < 0.001) in
OVE26 mice. Moreover, at 450 days OVE26 mice showed
118
TEIKEN ET AL.
TABLE 2. Mean glomerular volume, podocyte nuclear density, and podocyte number per glomerulus
in FVB and OVE26 mice 6 SEMa
VG: mean glomerular volume
3105mm3
Age (days)
60
150
450
a
NV: mean podocyte nuclear
density 31024/mm3
N: mean podocyte number per
glomerulus
FVB
OVE26
FVB
OVE26
FVB
OVE26
2.09 6 0.16
2.27 6 0.21
3.20 6 0.50
2.48 6 0.13*
5.24 6 0.47*
5.80 6 0.47*
7.29 6 0.94
7.84 6 1.33
5.19 6 1.14
5.92 6 1.26
3.19 6 0.31*
1.57 6 0.26*
151.33 6 19.79
172.85 6 23.77
164.15 6 26.52
147.09 6 30.68
165.35 6 14.78
82.56 6 12.98*
Asterisks indicate that comparisons between FVB and OVE26 mice show statistical significance (P < 0.05).
Fig. 3. A–C: Mean glomerular volume (A), mean podocyte nuclear density (B), and mean podocyte
number per glomerulus (C) 6 SEM in normal (FVB) and transgenic diabetic (OVE26) mice. Normal mice
are represented by open bars; diabetic mice are represented by filled bars. Asterisks indicate that comparisons between FVB and OVE26 mice show statistical significance (P < 0.05).
231% fewer podocyte nuclei/mm3 of glomerular tissue
than age-matched controls (Table 2; Fig. 3B).
Mean podocyte number per glomerulus (N).
For aging FVB mice, the mean N remained fairly constant (151 at 60 days, 172 at 150 days, and 164 at
450 days; Table 2; Fig. 3C). Of interest, N values in
OVE26 animals were similar to FVB mice at both 60 days
of age and 150 days of age. However, a substantial reduction (100%) occurred between 150 days and 450 days
when the mean N in the oldest OVE26 mice was reduced
to 83 (56% of 60 day values).
When average N was compared in FVB and OVE26
mice, controls and diabetics were similar at 60 days and
150 days. However, at 450 days, N was significantly less
(99%; P 5 0.005) in diabetic mice than in age-matched
controls (Table 2; Fig. 3C).
DISCUSSION
The OVE26 transgenic diabetic mouse is particularly
interesting because, despite substantial pancreatic bcell–specific damage (Epstein et al., 1989, 1992) and
blood glucose levels frequently exceeding 600 mg/dl
(Zheng et al., 2004), insulin secretion is sufficient to
allow these mice to live as long as 450 days without
treatment. Significantly, these mice show numerous
PODOCYTE MORPHOMETRY IN DIABETIC MICE
morphological features that parallel chronic complications of DN in humans (Carlson et al., 1997, 2003;
Zheng et al., 2004). Moreover, they are prone to early
and frequently severe microalbuminuria and the major
blood pressure effect is hypertension (Zheng et al.,
2004). On the contrary, throughout their life-span, FVB
mice show no significant functional or morphological
complications considered characteristic of DN (Carlson
et al., 1997, 2003; Zheng et al., 2004), despite their tendency toward blood glucose levels that could be considered hyperglycemic in human (Zheng et al., 2004).
Numerous reports and reviews suggest that podocytes
may be major players in the development of DN (Marshall, 2005; Shankland, 2006) and that podocyte injury
or loss may be directly or indirectly related to proteinuria. In the current study, we attempted to take advantage of the unique nephropathic features of diabetic
OVE26 mice in an effort to compare their mean values
of VG, NV and N, with those in age-matched FVB control
mice.
In patients, DN is characterized by renal and glomerular hypertrophy (Viberti et al., 1994). In the current
study, renal hypertrophy was evident in OVE26 mice by
150 days of age when kidney weights exceeded controls
by approximately 81%. Similar data were reported by
Zheng and coworkers (2004), who showed that at 5
months, kidney weights in OVE26 mice were 97%
greater than those from controls. Both studies showed
that body weights in diabetics and age-matched controls
were not significantly different.
In the current study, visual inspection of LM sections
of renal cortex in OVE26 animals showed glomerular
profiles that were larger in diameter than in agematched controls. This finding was confirmed by morphometric analyses carried out by the method of Weibel
and Gomez (Weibel and Gomez, 1962; Weibel, 1979) as
described by Lane and coworkers (1992) who demonstrated that when at least 15 glomerular profiles were
analyzed and adequate correction factors were applied,
reliable estimates of VG could be determined. Using this
method, we showed that at 60, 150, and 450 days, VG
was significantly increased in OVE26 mice over agematched controls. This finding was not unexpected as
glomerular hypertrophy is a consistent feature of both
early and late diabetic glomerulopathy (Osterby and
Gundersen, 1975, 1977). Moreover, using similar techniques on periodic acid-Schiff–stained paraffin sections,
Zheng and associates (2004) showed that glomerular volumes in OVE26 mice at 2.5, 6, and 10 months of age
were significantly enlarged relative to FVB control mice.
Because number of podocytes per glomerulus (N) was
a goal of the current studies and LM sections could not
be resolved sufficiently to quantify podocytes unequivocally, low-magnification TEM sections of entire glomerular profiles were used to determine NV, and ultimately N
was calculated for each glomerulus studied. Several
methods were available for determining NV, including
the disector method (Sterio, 1984), which some consider
to be the gold standard technique. However, this procedure required exhaustive sectioning of each glomerulus
and was not practical for our investigation. Because
TEM images were required and several investigators
(Steffes et al., 2001; White and Bilous, 2004) indicated
that the results using the disector method were not statistically different than those generated by the single-
119
section Weibel and Gomez (Weibel and Gomez, 1962;
Weibel, 1979) technique, provided the data were generated from electron micrographs, the latter method was
chosen. As a caveat, however, it should be pointed out
that a recent elegant light microscopic cell counting
study (Basgen et al., 2006) showed that the Weibel and
Gomez (Weibel and Gomez, 1962; Weibel, 1979) counting
method overestimated total glomerular cell counts by
10%.
Our data showed that, at 150 days and 450 days of
age, podocyte NV in OVE26 mice was significantly
reduced (146% and 231%, respectively) relative to
age-matched controls. Similar studies have not been carried out previously on OVE26 mice, so no direct comparisons can be made. Nevertheless, it has been well documented that podocyte numbers are reduced in human
(Pagtalunan et al., 1997; Shirato et al., 2001; Steffes
et al., 2001; White et al., 2002; Dalla Vestra et al., 2003)
and experimental (Petermann et al., 2004; Susztak
et al., 2006) DN. Although the precise mechanisms by
which podocytes are lost have not been elucidated, it has
been shown that glucose-induced apoptosis plays a role
in the process (Susztak et al., 2006). Of interest, however, it is likely that not all lost podocytes die, as viable
podocytes have been recovered in the urine of streptozotocin-induced diabetic rats (Petermann et al., 2004) and
human subjects with kidney disease (Vogelmann et al.,
2003). Regardless of the pathogenetic mechanisms it is
clear that, in human DN, podocyte loss is significant
and on average approximately 20% of podocytes are lost
within the first few years of the disease (Steffes et al.,
2001).
In the current study, podocyte NV was significantly
reduced in diabetic mice at 150 and 450 days of age.
However, NV was calculated as one step in determining
average N, and, although at 450 days N was significantly reduced (99%) in OVE26 animals compared
with normal mice, at 60 and 150 days, N was nearly
identical in diabetic and controls. These results were
somewhat unexpected as it has been shown that, in DN
patients (Pagtalunan et al., 1997; Shirato et al., 2001;
Steffes et al., 2001; White et al., 2002; Dalla Vestra
et al., 2003) and in several diabetic animal models
(Petermann et al., 2004; Susztak et al., 2006), N is generally reduced. It should be pointed out, however, that
in the study by White and coworkers (2002), there was
no reduction in podocyte numbers at baseline, and only
after 3 yr was there a significant podocyte loss, suggesting that this change can occur later in the disease.
We are intrigued by the significant reductions in NV
in 150- and 450-day-old diabetic mice and its variable
affect on N, which is necessarily directly related to concomitant increases in VG in these animals. In fact,
because N is dependent upon VG and NV, which are
inversely related in OVE26 and control mice, it is not
surprising that diabetics and age-matched controls show
relatively small differences in N.
An important question is thus raised: Is podocyte NV
or N the more functionally relevant factor? In this
regard, it must be pointed out that a decrease in NV can
be a result of (1) increased VG, (2) an absolute decrease
in N, or (3) a combination of both factors. Because a
decrease in N is not necessary in order for a decrease in
NV to occur, it is possible that NV may be the most relevant (and independent) data point in determining
120
TEIKEN ET AL.
glomerular function. Similar conclusions are expressed
by Dalla Vestra and coworkers (2003) who emphasize
the significance of NV and point out that N does not provide important information on glomerular architecture
such as how many cells may be available to cover the peripheral GBM in a large or small glomerular tuft. Further support for this position is derived from human
studies in which decreased podocyte NV correlates with
increased albumin excretion rates, suggesting a link
between reduced NV and permeability to albumin (Dalla
Vestra et al., 2003).
We recently showed that OVE26 mice exhibit pronounced UAE early in life and that it increases progressively with age (Zheng et al., 2004). Moreover, the data
indicate that, although the proteinuria is variable within
animal groups, by 9 months, average UAE exceeds 15
mg/24 hr. It seems possible, therefore, that the changes
in NV and N shown in the current study at 5 and 15
months may reflect podocyte losses that correlate with
the degree of increased proteinuria. Additional support
for this concept is offered in a recent transgenic rat
study that demonstrated a quantitative relationship
between the degree of podocyte depletion and the development of proteinuria and glomerulosclerosis (Wharram
et al., 2005).
It should be pointed out that the effects of blood pressure changes in OVE26 mice might be related to podocyte damage and loss. In this regard, it has been shown
that podocytes respond to injury by increased production
of extracellular matrix, detachment, and hypertrophy
(Kriz, 1996; Barisoni et al., 2000), and the latter is noted
specifically in response to stretch and mechanical stress
(Petermann et al., 2005). Because the primary blood
pressure effect in OVE26 mice is hypertension (Zheng
et al., 2004), it seems possible that the increased vasoactivity in this model may be linked to podocyte hypertrophy and reduced NV in late stages of life.
In conclusion, our data show that, in OVE26 transgenic diabetic mice, podocyte density and number
decrease significantly with age. Because these changes
occur relatively late in the course of the disease, it
seems possible that they may be a consequence of early
molecular diabetic nephropathic sequelae. Moreover,
because podocytes apparently do not divide in vivo (Kriz,
1996), they have little or no chance for cell proliferation
in response to injury. Accordingly, the observed reduction in podocyte numbers may be largely irreversible
and may play a role in sustaining the well-known
increased permeability of the blood–urine barrier in late
stages of diabetic renal decompensation.
ACKNOWLEDGMENTS
The authors thank Dr. Kap Lee for superb help with
animal health care and surgery, and Dr. Patrick Carr for
expert assistance with the statistical methods. E.C.C.
was funded by the North Dakota Lions Foundation;
P.N.E. was funded by a PHS grant and a JDRF grant;
and S.Z. was funded by a JDRF grant.
LITERATURE CITED
Adler S. 1994. Structure-function relationships associated with
extracellular matrix alterations in diabetic glomerulopathy. J Am
Soc Nephrol 5:1165–1172.
Asanuma K, Mundel P. 2003. The role of podocytes in glomerular
pathology. Clin Exp Nephrol 7:255–259.
Barisoni L, Mokrzycki M, Sablay L, Nagata M, Yamase H, Mundel
P. 2000. Podocyte cell regulation and proliferation in collapsing
glomerulopathies. Kidney Int 58:137–143.
Basgen JM, Nicholas SB, Mauer M, Rozen S, Nyengaard JR. 2006.
Comparison of methods for counting cells in the mouse glomerulus. Nephron Exp Nephrol 103:E139–E148.
Carlson EC, Audette JL, Klevay LM, Nguyen H, Epstein PN. 1997.
Ultrastructural and functional analyses of nephropathy in calmodulin-induced diabetic transgenic mice. Anat Rec 247:9–19.
Carlson EC, Audette JL, Veitenheimer NJ, Risan JA, Laturnus DI,
Epstein PN. 2003. Ultrastructural morphometry of capillary basement membrane thickness in normal and transgenic diabetic
mice. Anat Rec 271A:332–341.
Dalla Vestra M, Masiero A, Roiter AM, Saller A, Crepaldi G, Fioretto P. 2003. Is podocyte injury relevant in diabetic nephropathy?
Studies in patients with type 2 diabetes. Diabetes 52:1031–1035.
Epstein PN, Overbeek PA, Means AR. 1989. Calmodulin-induced
early-onset diabetes in transgenic mice. Cell 58:1067–1073.
Epstein PN, Ribar TJ, Decker GL, Yaney G, Means AR. 1992. Elevated b-cell calmodulin produces a unique insulin secretory defect
in transgenic mice. Endocrinology 130:1387–1393.
Goode NP, Shires M, Crellin DM, Aparicio SR, Davison AM. 1995.
Alterations of glomerular basement membrane charge and structure in diabetic nephropathy. Diabetologia 38:1455–1465.
Karnovsky MJ. 1965. A formaldehyde-glutaraldehyde fixative of
high osmolality for use in electron microscopy. J Cell Biol
27:137A–138A.
Kriz W. 1996. Progressive renal failure—inability of podocytes to
replicate and the consequences for development of glomerulosclerosis. Nephrol Dial Transplant 11:1738–1742.
Kriz W, Gretz N, Lemley KV. 1998. Progression of glomerular diseases: is the podocyte the culprit? Kidney Int 54:687–697.
Lane PH, Steffes MW, Mauer M. 1992. Estimation of glomerular
volume: a comparison of four methods. Kidney Int 41:1085–1089.
Macconi D, Bonomelli M, Benigni A, Plati T, Sangalli F, Longaretti L,
Conti S, Kawachi H, Hill P, Remuzzi G, Remuzzi A. 2006. Pathophysiologic implications of reduced podocyte number in a rat model
of progressive glomerular injury. Am J Pathol 168:42–54.
Marshall SM. 2005. The podocyte: a major player in the development of diabetic nephropathy? Horm Metab Res 37:9–16.
Mifsud SA, Allen TJ, Bertram JF, Hulthen UL, Kelly DJ, Cooper
ME, Wilkinson-Berka JL, Gilbert RE. 2001. Podocyte foot process
broadening in experimental diabetic nephropathy: amelioration
with renin-angiotensin blockade. Diabetologia 44:878–882.
Osterby R, Gundersen HJ. 1975. Glomerular size and structure in
diabetes. I. Early abnormalities. Diabetologia 11:225–229.
Osterby R, Gundersen HJ. 1977. Glomerular size and structure in
diabetes. I. Late abnormalities. Diabetologia 13:43–48.
Osterby R, Bangstad H-J, Nyberg G, Rudberg S. 2001. On glomerular structural alterations in type-1 diabetes. Virchows Arch
438:129–135.
Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers
BD, Rennke HG, Coplon NS, Sun L, Meyer TW. 1997. Podocyte
loss and progressive glomerular injury in type II diabetes. J Clin
Invest 99:342–348.
Pavenstädt H, Kriz W, Kretzler M. 2003. Cell biology of the glomerular podocyte. Physiol Rev 83:253–307.
Petermann AT, Pippin J, Krofft R, Blonski M, Griffin S, Durvasula
R, Shankland SJ. 2004. Viable podocytes detach in experimental
diabetic nephropathy: potential mechanism underlying glomerulosclerosis. Neph Exp Nephrol 98:E144–E123.
Petermann AT, Pippin J, Durvasula R, Pichler R, Hiromura K,
Monkawa T, Couser WG, Shankland SJ. 2005. Mechanical stretch
induces podocyte hypertrophy in vitro. Kidney Int 67:157–166.
Rincon-Choles H, Vasylyeva Tl, Pergola PE, Bhandari B, Bhandari
K, Zhang JH, Wang W, Gorin Y, Barnes JL, Abboud HE. 2006.
ZO-1 expression and phosphorylation in diabetic nephropathy.
Diabetes 55:894–900.
Shankland SJ. 2006. The podoctye’s response to injury: role in proteinuria and glomerulosclerosis. Kidney Int 69:2131–2147.
PODOCYTE MORPHOMETRY IN DIABETIC MICE
Shirato I, Hishiki T, Tomino Y. 2001. Podocyte loss and progression
of diabetic nephropathy. Contrib Nephrol 134:69–73.
Steffes MW, Osterby R, Chavers B, Mauer SM. 1989. Mesangial
expansion as a central mechanism for loss of kidney function in
diabetic patients. Diabetes 38:1077–1081.
Steffes MW, Schmidt D, McCrery R, Basgen JM. 2001. Glomerular
cell number in normal subjects and in type 1 diabetic patients.
Kidney Int 59:2104–2113.
Sterio DC. 1984. The unbiased estimation of number and sizes of
arbitrary particles using the disector. J Microscopy 134:127–136.
Susztak K, Raff AC, Schiffer M, Bottinger EP. 2006. Glucoseinduced reactive oxygen species cause apoptosis of podocytes and
podocyte depletion at the onset of diabetic nephropathy. Diabetes
55:225–233.
Viberti G, Wiseman MJ, Pinto JR, Messent J. 1994. Diabetic nephropathy. In: Kahn CR, Weir GC, editors. Joslin’s diabetes mellitus. 13th ed. Philadelphia: Lea and Febiger. p 691–737.
Vogelmann SU, Nelson WJ, Myers BD, Lemley KV. 2003. Urinary
excretion of viable podocytes in health and renal disease. Am J
Physiol Renal Physiol 285:F40–F48.
Weibel ER. 1979. Stereological methods. Vol. 1. Practical methods
for biological morphometry. London: Academic Press. p 44–45.
Weibel ER, Gomez DM. 1962. A principle for counting tissue structures on random sections. J Appl Physiol 17:343–348.
121
Wharram BL, Goyal M, Wiggins JE, Sanden SK, Hussain S,
Filipiak WE, Saunders TL, Dysko RC, Kohno K, Holzman LB,
Wiggins RC. 2005. Podocyte depletion causes glomerulosclerosis:
diphtheria toxin-induced podocyte depletion in rats expressing
human diphtheria toxin receptor transgene. J Am Soc Nephrol
16:2941–2952.
White KE, Bilous RW. 2004. Estimation of podocyte number: a comparison of methods. Kidney Int 66:663–667.
White KE, Bilous RW, Marshall SM, El Nahas M, Remuzzi G, Piras
G, De Cosmo S, Viberti G. 2002. Podocyte number in normotensive type I diabetic patients with albuminuria. Diabetes 51:3083–
3089.
Wolf G, Chen S, Ziyadeh FN. 2005. From the periphery of the glomerular capillary wall toward the center of disease. Podocyte
injury comes of age in diabetic nephropathy. Diabetes 54:1626–
1634.
Yu D, Petermann A, Kunter U, Rong S, Shankland SJ, Floege J.
2005. Urinary podocyte loss is a more specific marker of ongoing
glomerular damage than proteinuria. J Am Soc Nephrol 16:1733–
1741.
Zheng S, Noonan WT, Metreveli NS, Coventry S, Kralik PM, Carlson EC, Epstein PN. 2004. Development of late-stage diabetic
nephropathy in OVE26 diabetic mice. Diabetes 53:3248–
3257.
Документ
Категория
Без категории
Просмотров
0
Размер файла
335 Кб
Теги
loss, mice, podocyte, ove26, aging, diabetic
1/--страниц
Пожаловаться на содержимое документа