Kuze 1997
Kuze 1997
Kuze 1997
SUMMARY
Glomerular accumulation of the extracellular matrix (ECM) with subsequent sclerosis is a common finding in most progressive renal
diseases. Recently MSW (Mouse South Western) protein was cloned by its ability to bind the bidirectional promoter of the collagen IV
genes. This protein was also reported as the large subunit of the DNA replication complex A1, as well as the promoter binding protein
of corticotropin-releasing hormone and the angiotensinogen gene. To investigate the mechanism of accumulation of the ECM as it relates
to glomerular cellular events, the expression of MSW protein was studied in the remnant kidney model. Progressive expression of MSW
protein was found in the glomerular sclerotic lesion at week 4 and at later time points after renal ablation. The expression of proliferating
cell nuclear antigen (PCNA) and type IV collagen was also correlated with the expression of MSW protein by immunofluorescence.
RNA dot blot analysis also showed that the expression of MSW mRNA was increased at week 7 in association with the augmented
expression of type IV collagen. These results, taken together, suggest that MSW protein plays an important role in the regulation of type
IV collagen gene expression in vivo and may contribute to glomerular cell proliferation and the development of glomerulosclerosis.
? 1997 by John Wiley & Sons, Ltd.
injury. These findings indicate that the regulatory was graded semiquantitatively as follows: 0, no prolifer-
mechanism of this protein in vivo is essential to the ation of glomerular cell nuclei (less than 80 nuclei per
understanding of the pathological link between glomerulus); 1+, little proliferation of glomerular cell
glomerulosclerosis and glomerular cell proliferation and nuclei (about 100 nuclei per glomerulus); 2+, mild pro-
for clarifying the critical pathogenetic mechanisms for liferation of glomerular cell nuclei (about 120 nuclei per
glomerular injuries. However, the promoter binding glomerulus); 3+, moderate proliferation of glomerular
protein contributing to the expression of ECM genes cell nuclei (about 140 nuclei per glomerulus); 4+, signif-
and the proliferation of glomerular cells has not been icant proliferation of glomerular cell nuclei (more than
reported. Here we describe the augmented expression of 160 nuclei per glomerulus). Sections of the glomerular
MSW protein in parallel with the expression of type IV tuft, in which cross-sections had a very low cellularity
collagen and the severity of sclerotic lesions in the (arbitrarily defined as less than 50 cells per cross-
progressive glomerulosclerosis model. We also show a section), which are likely to represent tangential sections,
strong correlation between expression of MSW protein were excluded from the analysis. For the grade of
and cell proliferation in vivo. glomerulosclerosis and glomerular cell proliferation,
cross-sections of more than 30 consecutive cortical
glomeruli containing more than 30 discrete capillary
MATERIALS AND METHODS segments were evaluated by three observers, who were
unaware of the origin of the sections. Mean values were
Disease model and experimental protocol calculated.
Male Wistar rats (173 males from Shimizu Exper-
imental Material Corp., Japan) weighing 180–250 g were
Immunofluorescence staining
studied. Animals were fed standard rat chow and water.
In 116 rats (operation group), an infarction of approxi- For the immunofluorescence microscopic study, 4 ìm
mately two-thirds of the left kidney was accomplished thick cryostat sections of kidney were fixed in cold
by ligating the posterior and one or two anterior acetone for 5 min, washed with phosphate-buffered
branches of the main renal artery in combination saline (PBS) three times for 10 min each, and incubated
with right nephrectomy performed under sodium with 10 per cent goat serum in PBS for 30 min. The
pentobarbital intraperitoneal anaesthesia (50 mg/kg) sections were incubated with rabbit anti-type IV col-
(Nembutal; Dinabott Corporation, Osaka, Japan).11 In lagen antibody (HK681),13 rabbit anti-MSW anti-
the control group (sham) of 57 rats, a sham operation body,14 or mouse monoclonal anti-PCNA antibody
was performed without destruction of renal tissue. Post- (Oncogene Science Inc., NY, U.S.A.) for 30 min. They
operative urinary protein excretion, urinary creatinine, were washed with PBS and reacted with biotinylated
and serum creatinine values were determined at weeks 4, goat anti-rabbit IgG or biotinylated mouse IgG for 30
7, and 10 from randomly selected animals of both min at room temperature (TAGO, Inc., CA, U.S.A.).
groups. They were then washed and incubated with FITC con-
jugated with streptavidin (Zymed Laboratories, Inc.,
CA, U.S.A.) for 30 min. After final washing in PBS,
Renal morphology
they were mounted and observed by epifluorescence
Tissue for light microscopy was fixed in methyl microscopy (Nikon, Tokyo, Japan). The fluorescence
Carnoy’s solution and embedded in paraffin. Four- intensity of glomeruli was graded semiquantitatively as
micrometre sections were stained with haematoxylin and follows: 0, indistinguishable from control; 1, <25 per
eosin, periodic acid Schiff (PAS), and periodic silver cent of the tuft showing fluorescence; 2, 25–50 per cent;
methenamine. The percentage of glomeruli exhibiting 3, 50–75 per cent; and 4, >75 per cent.
focal or global glomerulosclerosis was determined.4
Glomerulosclerosis was characterized by an increase in
glomerular ECM, collapse and obliteration of capillary Preparation of glomerular RNA and RNA dot blot
lumina, and accumulation of hyaline in association with analysis of type IV collagen and MSW gene expression
adhesion to Bowman’s capsule. A semiquantitative score Glomeruli were isolated by differential sieving. Total
was used to evaluate the degree of glomerular sclerosis, RNA was extracted from isolated glomeruli from five
described by Raij et al.12 More than 30 glomeruli (n) separate groups of six operated and three sham-operated
were examined independently and the lesions were rats at weeks 4 and 7 by the method described by
graded from 0 to 4+, according to the percentage of Chomczynski and Sacchi.15 Glomerular mRNA levels
glomerulosclerosis. Involvement of less than 25 per cent for pools of kidneys from experimental or control ani-
of the glomerulus was regarded as a 1+ lesion, 25 to less mals were quantified by dot blot analysis prepared with
than 50 per cent 2+, 50 to less than 75 per cent 3+, and 1, 2, or 5 ìg of each RNA sample and control RNA.
75–100 per cent 4+. n1 was the number of glomeruli Aliquots were applied to wells with 1/3 serial dilutions
showing a 1+ lesion, n2 2+, n3 3+, and n4 4+, respect- and blotted onto nylon filters (Convertible Nylon
ively. The sclerosis index was calculated by the following Membranes, BRL, U.S.A.). Filters were cross-linked by
formula: [(1#n1)+(2#n2)+(3#n3)+(4#n4)]/n#100.12 short-wave UV. Prehybridization was performed for
Total glomerular cellularity was evaluated by counting 2 h at 42)C in 5# SSPE (20# SSPE: 3·6 sodium
the total number of nuclei per glomerulus. For the evalu- chloride, 0·2 sodium phosphate, 20 m EDTA;
ation of glomerular proliferating cells, each glomerulus pH 7·4), 5# Denhardt’s (100# Denhardt’s: 2 per cent
? 1997 by John Wiley & Sons, Ltd. , . 183: 16–23 (1997)
18 K. KUZE ET AL.
Table I—Basic characteristics of the renal ablation model: serum creatinine, urinary protein excretion, sclerosis index, and grade of
glomerular proliferating cells in rats after 5/6 nephrectomy (ope) or sham-operated rats (sham)
Ficoll 400, 2 per cent polyvinylpyrrolidone, 2 per cent were normalized for equivalent amounts of 18S ribo-
bovine serum albumin; BSA), 10 mg/ml salmon sperm somal RNA per dot to confirm equal loading of RNA.
DNA, 0·5 per cent sodium dodecyl sulphate (SDS), and The concentration of the probe was in excess over
50 per cent formamide. Membranes were hybridized that on the filter and the autoradiograph was not
with 40 ng of more than 1#109 cpm/mg probe with 2 ml overexposed.
of hybridization buffer (5# SSPE, 2# Denhardt’s,
10 mg/ml salmon sperm DNA, 0·5 per cent SDS, 50 per
cent formamide) overnight at 42)C. After hybridization, Miscellaneous measurements
membranes were washed twice with 5# SSC, 0·5 per Serum creatinine was measured using picric acid
cent SDS for 5 min at 65)C and in 0·1# SSC, 1 per cent (Creatinine Test Wako, Wako Pure Chemical Industries,
SDS at 50)C once for 30 min. Finally, they were washed Osaka, Japan) and urine protein excretion was measured
with 2# SSC once at room temperature for 5 min. The by a dye binding assay using a BSA standard (Bio-Rad
membranes were exposed to Konica X-ray film with protein assay kit, Bio-Rad, Osaka, Japan).
intensifying screens at "70)C. The cDNA probes
used for this analysis were as follows: (1) MSW cDNA:
a 1·8 kb XhoI fragment of murine MSW cDNA in Statistical analysis
Bluescript was used for the detection of the rat MSW The data were expressed as the mean&standard error
transcript; (2) type IV collagen cDNA (p1234): about and were evaluated statistically by analysis of variance
0·8 kb EcoRI–HindIII fragment of murine type IV col- or a non-parametric test (Mann–Whitney analysis).
lagen cDNA was used to detect the rat type IV collagen
transcript;16 (3) 18S ribosomal RNA: pN29III (Rn18s)
was used to detect 18S ribosomal RNA (American Type RESULTS
Culture Collection, MD, U.S.A.).17 All probes were
labelled with [á-32P]-deoxycytidine 5-triphosphate (3000 Progressive renal insufficiency, proteinuria, glomerular
Ci/mmol; Amersham Corp., Arlington Heights, IL, cell proliferation, and glomerulosclerosis develop after
U.S.A.) by a random primer extension kit (Pharmacia, 5/6 nephrectomy
U.K.). Autoradiograms were analysed by linear Following the 5/6 nephrectomy, a significant rise of
densitometry. All dot blot analyses were repeated two to serum creatinine did not occur until week 10. Pathologi-
three times with glomerular RNA preparations obtained cal proteinuria (>mean proteinuria of sham-operated
from different sets of animals. Some membranes were rats+2 standard deviations) was present at week 4 and
rehybridized with additional probes (up to a maximum remained until week 10 in the operation group (Table I),
of two times). but pathological proteinuria was absent in the sham
group. By light microscopy, at week 4, PAS staining
revealed focal and segmental glomerulonephritis with
Quantitation of MSW and type IV collagen mRNA less than 50 per cent of the glomeruli showing localized
For more accurate quantitation, densitometric segmental sclerosis and mesangial cell proliferation; the
analysis was performed using Imagemaster software remaining glomeruli showed only minor changes in the
(Pharmacia, Image Master TM, U.S.A.) on an IBM nephrectomized rats. As a week passed, much more
Personal System/V computer. A scan was made of a mesangial cell proliferation and ECM accumula-
series of samples. Contour OD#mm2, which is a histo- tion occurred with interstitial damage (Fig. 1). We
gram of the density of each contoured band in a determined the amount of glomerular cell proliferation
matched group, was calculated. Densitometry readings semiquantitatively by counting the total number of
? 1997 by John Wiley & Sons, Ltd. , . 183: 16–23 (1997)
TRANSCRIPTION FACTOR IN GLOMERULOSCLEROSIS 19
Fig. 1—Glomerular PAS staining of tissue obtained from rats with 5/6 nephrectomy at weeks 4, 7, and 10 after the operation (A, B, C) and rats at
week 10 after the sham operation (D)
nuclei per glomerulus. After the operation, glomerular week 1 and remained until week 10. This result was
cells showed biphasic proliferation at weeks 4 and 10. consistent with the grade of glomerular cell proliferation
We also calculated the sclerosis index at weeks 4, 7, and described above (Figs 2 and 3). We also calculated the
10. The presence of focal glomerulosclerosis was semiquantitative intensity of immunofluorescent stain-
noted in a minority of glomeruli at week 1 (data not ing of glomeruli in the nephrectomized rats chronologi-
shown), subsequent to progressive sclerotic changes cally, compared with the sham-operated rats. MSW
over time. No significant change was observed in the protein at week 10 increased approximately two-fold
sham-operated rats (Table I). compared with week 4 following 5/6 nephrectomy. Type
IV collagen at week 10 increased about two-fold com-
pared with week 1 after the operation. MSW protein was
Increased expression of MSW, PCNA, and type IV
thus induced by 5/6 nephrectomy in parallel to the
collagen protein during glomerular cell proliferation and
expression of type IV collagen. Interestingly, augmented
glomerulosclerosis
expression of MSW protein was associated with
Fluorescence staining showed remarkably increased glomerular cell proliferation and increased expression of
expression of MSW protein parallel to the expression of PCNA (Table II).
type IV collagen in nephrectomized rats as glomerulo-
sclerosis developed, but no increased expression was
observed in sham-operated rats (Figs 2 and 3). By Augmented expression of MSW mRNA correlated with
Western blot analysis, the intensity of the bands of the expression of type IV collagen mRNA
serially operated rats was also stronger than sham-
operated rats. This result indicated that rabbit anti- We investigated transcripts of the MSW and type IV
MSW antibody could bind specifically to the epitope of collagen genes in rat glomeruli by RNA dot blot analy-
MSW protein (data not shown). At week 1, increased sis. Augmented expression of MSW mRNA was
expression of type IV collagen and MSW protein was observed at week 7 in the nephrectomized rats, com-
observed. The expression of type IV collagen continued pared with the sham-operated rats. This increased
until week 10 and the protein was mainly localized to the expression was correlated to the induced expression of
mesangial area. The expression of PCNA increased from type IV collagen mRNA (shown in Fig. 4).
? 1997 by John Wiley & Sons, Ltd. , . 183: 16–23 (1997)
20 K. KUZE ET AL.
Fig. 2—Immunofluorescence glomerular staining for MSW protein (A), type IV collagen (B), and
PCNA (C) in rats at week 7 after 5/6 nephrectomy and in sham-operated rats at week 7 (D, E, F)
Densitometric analysis showed that MSW transcripts our method has previously been used many times and is
and type IV collagen transcripts increased approxi- simple and reliable. After the pathological episode of
mately 2·6-fold and about 4·3-fold at week 7 after 5/6 glomerular cell proliferation, the accumulation of
nephrectomy, respectively, compared with the sham- glomerular ECM occurs and is maintained for more
operated rats. than 10 weeks.
This study demonstrates the pathological role of
MSW, which has two distinct functions, both DNA
DISCUSSION replication and transcription of the type IV collagen
genes. We found that the increased level of MSW
The most extensively studied model of progressive correlated with high expression of type IV collagen
glomerulosclerosis leading to end-stage kidney disease is and the severity of glomerulosclerosis, and that the
the 5/6 nephrectomy model in rats.4,18 In this model, increased expression of MSW was also associated with
mesangial cell proliferation starts as early as 5 days after glomerular cell proliferation in vivo. These findings
nephrectomy and almost completely subsides at week suggest that MSW plays a pivotal role in regulating both
10. Although there remain many problems with count- cell proliferation and glomerulosclerosis, independent of
ing cells in glomeruli and with PCNA immunostaining, initial insults, and may contribute to the initiation of the
? 1997 by John Wiley & Sons, Ltd. , . 183: 16–23 (1997)
TRANSCRIPTION FACTOR IN GLOMERULOSCLEROSIS 21
Table II—Glomerular immunofluorescence staining scores observed for PCNA, type IV collagen, and MSW protein in 5/6
nephrectomized rats or sham-operated rats
Fig. 4—Dot blot analysis of total glomerular RNA obtained in rats at week 7 after 5/6 nephrectomy (O), and in sham-operated rats
(S) at week 7 for the expression of MSW mRNA, collagen IV mRNA, and 18S rRNA. Serial dilutions of RNAs were prepared
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