Original Paper Nephron Exp Nephrol 2014;126:183–190 DOI: 10.1159/000362555
Received: June 7, 2013 Accepted: March 25, 2014 Published online: July 3, 2014
Ischemia/Reperfusion of Unilateral Kidney Exaggerates Aging-Induced Damage to the Heart and Contralateral Kidney Junichiro Kato a, c Masaaki Nakayama a, b, d Wan-Jun Zhu a, d Takashi Yokoo c Sadayoshi Ito a, b a
Research Division of CKD and Dialysis, and b Center for Advanced Integrated Renal Science, Tohoku University Graduate School of Medicine, Sendai, c Department of Kidney and Hypertension, The Jikei University School of Medicine, Tokyo, and d Department of Kidney and Hypertension, Fukushima Medical University School of Medicine, Fukushima, Japan
Key Words Inflammation · Cardiovascular diseases · Macrophage infiltration · Fibrosis
Abstract Aims: We aimed to determine the impact of aging on ischemic acute kidney injury, especially in terms of the pathological mechanisms of kidney and heart crosstalk. Method: The effects of 45 min of unilateral ischemic reperfusion (IR) of the renal artery on the contralateral kidney and heart were histologically assessed in 7- and 40-week-old SD rats after 7 days. Results: Glomerular sclerosis, interstitial fibrosis and numbers of ED1 cells were significantly increased in the contralateral kidneys of the 40-, but not the 7-week-old rats. The numbers of ED1 cells in the heart significantly and similarly increased in both groups, but reactive fibrosis after IR was significant only in the 40-week-old rats. The exaggerated profibrotic response induced by aging seemed to be closely associated with the increased number of ED1 cells in the affected area. Conclusion: Aging could play a major role in exaggerating the pathological processes of inflammation to fibrosis in remote organs including the heart and the nonischemic kidney after IR stimulation of the unilateral kidney. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 1660–2129/14/1264–0183$39.50/0 E-Mail [email protected] www.karger.com/nee
Introduction
In parallel with the increasing numbers of elderly people in developed countries, more patients are presenting with acute kidney injury (AKI) [1, 2]. The development of AKI is associated with higher rates of mortality and worse long-term prognoses in patients with AKI accompanied by heart failure [3, 4] or ischemic coronary diseases [5, 6], or who have undergone coronary artery surgery [7, 8]. With respect to the clinical significance of ischemic AKI, the underlying pathological mechanisms of kidney-heart crosstalk, namely cardiorenal syndrome [9], and the influence of aging require clarification. Ischemic reperfusion (IR) stimulates leukocyte infiltration into the affected site and into remote organs [10], and neutrophils in the acute phase accumulate at sites of chronic inflammation followed by mononuclear cells [11]. Bilateral kidney IR in experimental models of AKI causes left ventricular dysfunction and neutrophil infiltration [12], and unilateral kidney IR enhances macrophage infiltration into the nonischemic contralateral kidney [13–16] and heart [13, 14]. These findings indicate that systemic inflammatory activation plays a critical role in response to the ischemic kidney. On the other hand, the immune function is impaired in aging individuals as Masaaki Nakayama, MD, PhD Fukushima Medical University School of Medicine 1 Hikarigaoka Fukushima (Japan) E-Mail masanaka @ fmu.ac.jp
glyoxal levels were determined by LC/MS as described [19]. Urinary protein was measured using the Quick Start bovine serum albumin standard set (Bio-Rad Laboratories, Hercules, Calif., USA). Urinary thiobarbituric acid-reactive substances were measured using lipid peroxidation assays, and plasma creatinine and blood urea nitrogen were determined using an autoanalyzer (Beckman Coulter, Fullerton, Calif., USA).
Unilateral kidney (left) Ischemic reperfusion (45 min)
Right kidney
Left kidney
Non-IR kidney
IR kidney
Fig. 1. Protocol for study of unilateral kidney IR. The left renal artery of test rats was clamped for 45 min under anesthesia and then reperfused.
indicated by a decrease in the number of naïve T cells, and increased production of proinflammatory chemokines [17, 18], and these are clinical characteristics that might enhance the development of prolonged inflammation. Thus, elderly persons with AKI might be more prone to an exaggerated vicious cardiorenal cycle. The present study examines the effect of unilateral ischemic AKI in 7and 40-week-old SD rats to determine the effects of aging.
Histological Assessment Kidney and heart sections were assessed by Masson’s trichrome staining to determine renal injury and cardiac fibrosis. Kidney and heart tissues were immediately fixed in 95% ethanol overnight, followed by 100% ethanol overnight and then embedded in paraffin. Sections (3 μm thick) mounted on glass slides were deparaffinized with xylene and ethanol and then immunohistochemically stained with monoclonal antibodies against ED1 (Serotec, Oxford, UK), CD163 (M2; Leica Biosystems, Wetzlar, Germany), and osteopontin (OPN; Santa Cruz Biotechnology, Santa Cruz, Calif., USA). The slides were then incubated overnight at 4 ° C. The glomerular matrix was semiquantified in 50 randomly selected glomeruli. The ratios of glomeruli occupied by mesangial matrix were estimated and scored as 0 (normal), 1, 3 or 4 (75% of each glomerulus, respectively). Glomerular injury scores (GIS) were generated using the formula:
[(0 × n0) + (1 × n1) + (2 × n2) + (3 × n3) + (4 × n4)]/50. Parameters including the area of fibrosis, OPN, ED1, and CD163 were semiquantified in five randomly selected areas of the outer medulla of kidney or heart tissues (magnification, ×200) at largest vertical sectioned area of the left ventricle. The ratios (%) of each area with sclerotic or fibrotic changes (blue Masson’s trichrome stain) or positive (immune staining) were determined using Image J software (National Institutes of Health, Bethesda, Md., USA). Cells that were positive for ED1 and CD163 were counted in all selected areas.
Materials and Methods Animals and Protocols Six-week-old male Dahl salt-sensitive rats housed in a room under controlled temperature and humidity with 12-hour light/ dark cycles were fed with a 0.5% salt diet during the study period. The left renal artery of 7-week-old rats was clamped under intraperitoneal phenobarbital for 45 min and reperfused (fig. 1), and then the systemic effect of IR was determined by plasma analysis and histological assessment of groups of 6 rats at 3 h and at 1, 3, 7 and 14 days after IR. Based on the findings of this process, the effects of aging were histologically determined in 7- and 40-week-old rats at 1 week after unilateral kidney IR or sham procedure (7-weekold rats, n = 10; 40-week-old rats, n = 6). Animal care and handling proceeded in accordance with our institutional guidelines for the care and use of laboratory animals, and the Animal Committee at Tohoku University School of Medicine approved the study protocols (No. 2011-390). Measurements Blood pressure was measured in the morning by the tail-cuff method using an MK2000A blood pressure monitor for mice and rats (Muromachi, Tokyo, Japan). Plasma levels of MCP-1, and TNF-α were determined using respective rat immunoassay kits (Invitrogen Corporation, Carlsbad, Calif., USA). Plasma methyl-
184
Nephron Exp Nephrol 2014;126:183–190 DOI: 10.1159/000362555
Statistical Analysis Data are expressed as means ± standard error of the mean and were analyzed using an independent t test or two-way repeatedmeasures analysis of variance. Differences between groups were considered significant at p < 0.05. All data were statistically analyzed using Sigmastat 3.5 software (Systat Software, Chicago, Ill., USA).
Results
Time Course Changes in Parameters after Unilateral Kidney IR Table 1 and figure 2 show time course changes in kidney injury parameters after unilateral kidney IR in the 7-week-old rats. Plasma creatinine levels transiently increased, but changes in proinflammatory cytokines did not significantly change (table 1). However, OPN expression increased over time, reaching a maximum at day 1 (fig. 2a), and the number of ED1-positive cells was maximal at day 7 (fig. 2b) in the IR kidney. Kato /Nakayama /Zhu /Yokoo /Ito
OPN-positive area (%)
3.0 2.5 2.0 1.5 1.0
0
Number of ED1-positive cells/HPF
7-week-old rats after unilateral kidney IR. * p < 0.05; vs. control kidney, † p < 0.05; vs. non-IR kidney or IR kidney. HPF = High power field.
†
*, †
0.5
a
Fig. 2. Time course changes in OPN (a) and numbers of ED1 cells (b) in kidneys of
Control kidney Non-IR kidney IR kidney
*
Day 1
3h
Day 3
Day 7
Day 14
2.5 2.0
*
1.5
*
1.0
* *
0.5
b
0
*
3h
Day 1
Day 3
Day 7
*, †
Day 14
Table 1. Time course of changes in kidney parameters from 3 h to 14 days after unilateral kidney IR in 7-week-
old SD rats
Cre, mg/dl MCP-1, pg/ml TNF-α, pg/ml MGO, nM FENa, %
Model
3h
Day 1
Day 3
Day 7
Day 14
Sham IR Sham IR Sham IR Sham IR Sham IR
0.17 ± 0.02 0.33 ± 0.02* 58.4 ± 7.4 113.0 ± 13.3 85.2 ± 11.7 166.0 ± 38.4 151 ± 21 125 ± 3 NA NA
0.21 ± 0.04 0.40 ± 0.08 72.6 ± 1.2 79.9 ± 0.8 49.3 ± 2.4 144.2 ± 69.0 133 ± 7 139 ± 3 NA NA
0.28 ± 0.07 0.33 ± 0.03 48.4 ± 0.9 58.6 ± 1.6 53.2 ± 3.6 58.0 ± 4.2 114 ± 3 121 ± 10 0.24 ± 0.09 0.34 ± 0.03
0.21 ± 0.02 0.30 ± 0.02 51.7 ± 1.1 54.0 ± 2.3 60.3 ± 1.0 76.2 ± 12.2 105 ± 3 146 ± 17 0.26 ± 0.02 0.33 ± 0.02
0.20 ± 0.00 0.23 ± 0.02 37.3 ± 10.3 39.8 ± 6.8 50.9 ± 7.4 69.0 ± 18.5 112 ± 3 160 ± 23 0.21 ± 0.01 0.23 ± 0.04
Data are shown as means ± SE. Cre = Creatinine; FENa = fractional excretion of urinary sodium; MGO = methylglyoxal; NA = not available. * p < 0.05 vs. sham.
Comparisons between the 7- and 40-Week-Old Rats after Unilateral Kidney IR Table 2 shows that mean body weight, blood pressure, serum creatinine, urinary albumin and thiobarbituric acid-reactive substances did not significantly differ among the 7- and 40-week-old rats with IR and sham-operated rats.
Histological and Semiquantitative Findings of Kidneys Figure 3a–f, j and k shows representative histological findings of kidneys from the sham and IR rats. Figure 4 shows semiquantitative histological changes in kidneys from the IR groups aged 7 and 40 weeks. Glomerular Injury Scores. The GIS of the IR kidney did not significantly differ in the 7-week-old rats, but sig-
Enhanced Inflammation after Ischemic Reperfusion by Aging
Nephron Exp Nephrol 2014;126:183–190 DOI: 10.1159/000362555
185
b
c
d
e
f
g
h
i
j
k
l
m
n
Fig. 3. Representative histological findings of the kidney and heart.
Shriveled cortical glomerulus in kidneys of 7-week-old sham-operated rats (a) is accompanied by mildly increased mesangial matrix (blue) in 40-week-old sham-operated rats (b) and by increased mesangial matrix with some adhesions in the contralateral kidney of 40-week-old IR rats (c). Interstitial fibrosis is faint (d) and mildly increased (e) in 7- and 40-week-old sham-operated rats and moderately increased with damaged tubules (f) in the contralat-
186
Nephron Exp Nephrol 2014;126:183–190 DOI: 10.1159/000362555
Color version available online
a
eral kidney of 40-week-old IR rats. Interstitial fibrosis is faint, mildly and moderately increased in the myocardium of 7-weekold (g) and 40-week-old sham-operated (h) and 40-week-old IR (i) rats. Representative immunostaining for OPN (j) and ED1 (k; arrows) in the outer medulla of the kidney and for ED1 in the myocardium (l; arrows). Immunostaining for ED1 (m; arrowheads) and CD163 (n; arrowheads) in the sequential slice specimen of the kidney.
Kato /Nakayama /Zhu /Yokoo /Ito
Control kidney Non-IR kidney IR kidney
15
# Fibrosis area (%)/HPF
1.5
GIS
1.0
0.5
a
0
7
b
40
#
2.0
1.0
0
c
Number of ED1-positive cells/HPF
OPN staining area (%)/HPF
3.0
+
7
40
# 10
5 # 0
7
15
#
10
5
+ +
0
#
7
d
Age (weeks)
40
40 Age (weeks)
Fig. 4. Semiquantitative analysis of histological changes in the kidney. Glomerular adhesion (a), interstitial fibrosis (b), OPN (c), and number of ED1-positive cells (d). # p < 0.05; vs. control kidney (a, c, d), and non-IR kidney (b), + p < 0.05; vs. control kidney (c, d).
Table 2. Comparison of parameters in
7-week-old rats
7- and 40-week-old SD rats at 7 days after unilateral IR
sham Body weight, g BP, mm Hg Cre, mg/l
IR
40-week-old rats p
234.3 ± 5.9 284.3 ± 1.9
Received: June 7, 2013 Accepted: March 25, 2014 Published online: July 3, 2014
Ischemia/Reperfusion of Unilateral Kidney Exaggerates Aging-Induced Damage to the Heart and Contralateral Kidney Junichiro Kato a, c Masaaki Nakayama a, b, d Wan-Jun Zhu a, d Takashi Yokoo c Sadayoshi Ito a, b a
Research Division of CKD and Dialysis, and b Center for Advanced Integrated Renal Science, Tohoku University Graduate School of Medicine, Sendai, c Department of Kidney and Hypertension, The Jikei University School of Medicine, Tokyo, and d Department of Kidney and Hypertension, Fukushima Medical University School of Medicine, Fukushima, Japan
Key Words Inflammation · Cardiovascular diseases · Macrophage infiltration · Fibrosis
Abstract Aims: We aimed to determine the impact of aging on ischemic acute kidney injury, especially in terms of the pathological mechanisms of kidney and heart crosstalk. Method: The effects of 45 min of unilateral ischemic reperfusion (IR) of the renal artery on the contralateral kidney and heart were histologically assessed in 7- and 40-week-old SD rats after 7 days. Results: Glomerular sclerosis, interstitial fibrosis and numbers of ED1 cells were significantly increased in the contralateral kidneys of the 40-, but not the 7-week-old rats. The numbers of ED1 cells in the heart significantly and similarly increased in both groups, but reactive fibrosis after IR was significant only in the 40-week-old rats. The exaggerated profibrotic response induced by aging seemed to be closely associated with the increased number of ED1 cells in the affected area. Conclusion: Aging could play a major role in exaggerating the pathological processes of inflammation to fibrosis in remote organs including the heart and the nonischemic kidney after IR stimulation of the unilateral kidney. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 1660–2129/14/1264–0183$39.50/0 E-Mail [email protected] www.karger.com/nee
Introduction
In parallel with the increasing numbers of elderly people in developed countries, more patients are presenting with acute kidney injury (AKI) [1, 2]. The development of AKI is associated with higher rates of mortality and worse long-term prognoses in patients with AKI accompanied by heart failure [3, 4] or ischemic coronary diseases [5, 6], or who have undergone coronary artery surgery [7, 8]. With respect to the clinical significance of ischemic AKI, the underlying pathological mechanisms of kidney-heart crosstalk, namely cardiorenal syndrome [9], and the influence of aging require clarification. Ischemic reperfusion (IR) stimulates leukocyte infiltration into the affected site and into remote organs [10], and neutrophils in the acute phase accumulate at sites of chronic inflammation followed by mononuclear cells [11]. Bilateral kidney IR in experimental models of AKI causes left ventricular dysfunction and neutrophil infiltration [12], and unilateral kidney IR enhances macrophage infiltration into the nonischemic contralateral kidney [13–16] and heart [13, 14]. These findings indicate that systemic inflammatory activation plays a critical role in response to the ischemic kidney. On the other hand, the immune function is impaired in aging individuals as Masaaki Nakayama, MD, PhD Fukushima Medical University School of Medicine 1 Hikarigaoka Fukushima (Japan) E-Mail masanaka @ fmu.ac.jp
glyoxal levels were determined by LC/MS as described [19]. Urinary protein was measured using the Quick Start bovine serum albumin standard set (Bio-Rad Laboratories, Hercules, Calif., USA). Urinary thiobarbituric acid-reactive substances were measured using lipid peroxidation assays, and plasma creatinine and blood urea nitrogen were determined using an autoanalyzer (Beckman Coulter, Fullerton, Calif., USA).
Unilateral kidney (left) Ischemic reperfusion (45 min)
Right kidney
Left kidney
Non-IR kidney
IR kidney
Fig. 1. Protocol for study of unilateral kidney IR. The left renal artery of test rats was clamped for 45 min under anesthesia and then reperfused.
indicated by a decrease in the number of naïve T cells, and increased production of proinflammatory chemokines [17, 18], and these are clinical characteristics that might enhance the development of prolonged inflammation. Thus, elderly persons with AKI might be more prone to an exaggerated vicious cardiorenal cycle. The present study examines the effect of unilateral ischemic AKI in 7and 40-week-old SD rats to determine the effects of aging.
Histological Assessment Kidney and heart sections were assessed by Masson’s trichrome staining to determine renal injury and cardiac fibrosis. Kidney and heart tissues were immediately fixed in 95% ethanol overnight, followed by 100% ethanol overnight and then embedded in paraffin. Sections (3 μm thick) mounted on glass slides were deparaffinized with xylene and ethanol and then immunohistochemically stained with monoclonal antibodies against ED1 (Serotec, Oxford, UK), CD163 (M2; Leica Biosystems, Wetzlar, Germany), and osteopontin (OPN; Santa Cruz Biotechnology, Santa Cruz, Calif., USA). The slides were then incubated overnight at 4 ° C. The glomerular matrix was semiquantified in 50 randomly selected glomeruli. The ratios of glomeruli occupied by mesangial matrix were estimated and scored as 0 (normal), 1, 3 or 4 (75% of each glomerulus, respectively). Glomerular injury scores (GIS) were generated using the formula:
[(0 × n0) + (1 × n1) + (2 × n2) + (3 × n3) + (4 × n4)]/50. Parameters including the area of fibrosis, OPN, ED1, and CD163 were semiquantified in five randomly selected areas of the outer medulla of kidney or heart tissues (magnification, ×200) at largest vertical sectioned area of the left ventricle. The ratios (%) of each area with sclerotic or fibrotic changes (blue Masson’s trichrome stain) or positive (immune staining) were determined using Image J software (National Institutes of Health, Bethesda, Md., USA). Cells that were positive for ED1 and CD163 were counted in all selected areas.
Materials and Methods Animals and Protocols Six-week-old male Dahl salt-sensitive rats housed in a room under controlled temperature and humidity with 12-hour light/ dark cycles were fed with a 0.5% salt diet during the study period. The left renal artery of 7-week-old rats was clamped under intraperitoneal phenobarbital for 45 min and reperfused (fig. 1), and then the systemic effect of IR was determined by plasma analysis and histological assessment of groups of 6 rats at 3 h and at 1, 3, 7 and 14 days after IR. Based on the findings of this process, the effects of aging were histologically determined in 7- and 40-week-old rats at 1 week after unilateral kidney IR or sham procedure (7-weekold rats, n = 10; 40-week-old rats, n = 6). Animal care and handling proceeded in accordance with our institutional guidelines for the care and use of laboratory animals, and the Animal Committee at Tohoku University School of Medicine approved the study protocols (No. 2011-390). Measurements Blood pressure was measured in the morning by the tail-cuff method using an MK2000A blood pressure monitor for mice and rats (Muromachi, Tokyo, Japan). Plasma levels of MCP-1, and TNF-α were determined using respective rat immunoassay kits (Invitrogen Corporation, Carlsbad, Calif., USA). Plasma methyl-
184
Nephron Exp Nephrol 2014;126:183–190 DOI: 10.1159/000362555
Statistical Analysis Data are expressed as means ± standard error of the mean and were analyzed using an independent t test or two-way repeatedmeasures analysis of variance. Differences between groups were considered significant at p < 0.05. All data were statistically analyzed using Sigmastat 3.5 software (Systat Software, Chicago, Ill., USA).
Results
Time Course Changes in Parameters after Unilateral Kidney IR Table 1 and figure 2 show time course changes in kidney injury parameters after unilateral kidney IR in the 7-week-old rats. Plasma creatinine levels transiently increased, but changes in proinflammatory cytokines did not significantly change (table 1). However, OPN expression increased over time, reaching a maximum at day 1 (fig. 2a), and the number of ED1-positive cells was maximal at day 7 (fig. 2b) in the IR kidney. Kato /Nakayama /Zhu /Yokoo /Ito
OPN-positive area (%)
3.0 2.5 2.0 1.5 1.0
0
Number of ED1-positive cells/HPF
7-week-old rats after unilateral kidney IR. * p < 0.05; vs. control kidney, † p < 0.05; vs. non-IR kidney or IR kidney. HPF = High power field.
†
*, †
0.5
a
Fig. 2. Time course changes in OPN (a) and numbers of ED1 cells (b) in kidneys of
Control kidney Non-IR kidney IR kidney
*
Day 1
3h
Day 3
Day 7
Day 14
2.5 2.0
*
1.5
*
1.0
* *
0.5
b
0
*
3h
Day 1
Day 3
Day 7
*, †
Day 14
Table 1. Time course of changes in kidney parameters from 3 h to 14 days after unilateral kidney IR in 7-week-
old SD rats
Cre, mg/dl MCP-1, pg/ml TNF-α, pg/ml MGO, nM FENa, %
Model
3h
Day 1
Day 3
Day 7
Day 14
Sham IR Sham IR Sham IR Sham IR Sham IR
0.17 ± 0.02 0.33 ± 0.02* 58.4 ± 7.4 113.0 ± 13.3 85.2 ± 11.7 166.0 ± 38.4 151 ± 21 125 ± 3 NA NA
0.21 ± 0.04 0.40 ± 0.08 72.6 ± 1.2 79.9 ± 0.8 49.3 ± 2.4 144.2 ± 69.0 133 ± 7 139 ± 3 NA NA
0.28 ± 0.07 0.33 ± 0.03 48.4 ± 0.9 58.6 ± 1.6 53.2 ± 3.6 58.0 ± 4.2 114 ± 3 121 ± 10 0.24 ± 0.09 0.34 ± 0.03
0.21 ± 0.02 0.30 ± 0.02 51.7 ± 1.1 54.0 ± 2.3 60.3 ± 1.0 76.2 ± 12.2 105 ± 3 146 ± 17 0.26 ± 0.02 0.33 ± 0.02
0.20 ± 0.00 0.23 ± 0.02 37.3 ± 10.3 39.8 ± 6.8 50.9 ± 7.4 69.0 ± 18.5 112 ± 3 160 ± 23 0.21 ± 0.01 0.23 ± 0.04
Data are shown as means ± SE. Cre = Creatinine; FENa = fractional excretion of urinary sodium; MGO = methylglyoxal; NA = not available. * p < 0.05 vs. sham.
Comparisons between the 7- and 40-Week-Old Rats after Unilateral Kidney IR Table 2 shows that mean body weight, blood pressure, serum creatinine, urinary albumin and thiobarbituric acid-reactive substances did not significantly differ among the 7- and 40-week-old rats with IR and sham-operated rats.
Histological and Semiquantitative Findings of Kidneys Figure 3a–f, j and k shows representative histological findings of kidneys from the sham and IR rats. Figure 4 shows semiquantitative histological changes in kidneys from the IR groups aged 7 and 40 weeks. Glomerular Injury Scores. The GIS of the IR kidney did not significantly differ in the 7-week-old rats, but sig-
Enhanced Inflammation after Ischemic Reperfusion by Aging
Nephron Exp Nephrol 2014;126:183–190 DOI: 10.1159/000362555
185
b
c
d
e
f
g
h
i
j
k
l
m
n
Fig. 3. Representative histological findings of the kidney and heart.
Shriveled cortical glomerulus in kidneys of 7-week-old sham-operated rats (a) is accompanied by mildly increased mesangial matrix (blue) in 40-week-old sham-operated rats (b) and by increased mesangial matrix with some adhesions in the contralateral kidney of 40-week-old IR rats (c). Interstitial fibrosis is faint (d) and mildly increased (e) in 7- and 40-week-old sham-operated rats and moderately increased with damaged tubules (f) in the contralat-
186
Nephron Exp Nephrol 2014;126:183–190 DOI: 10.1159/000362555
Color version available online
a
eral kidney of 40-week-old IR rats. Interstitial fibrosis is faint, mildly and moderately increased in the myocardium of 7-weekold (g) and 40-week-old sham-operated (h) and 40-week-old IR (i) rats. Representative immunostaining for OPN (j) and ED1 (k; arrows) in the outer medulla of the kidney and for ED1 in the myocardium (l; arrows). Immunostaining for ED1 (m; arrowheads) and CD163 (n; arrowheads) in the sequential slice specimen of the kidney.
Kato /Nakayama /Zhu /Yokoo /Ito
Control kidney Non-IR kidney IR kidney
15
# Fibrosis area (%)/HPF
1.5
GIS
1.0
0.5
a
0
7
b
40
#
2.0
1.0
0
c
Number of ED1-positive cells/HPF
OPN staining area (%)/HPF
3.0
+
7
40
# 10
5 # 0
7
15
#
10
5
+ +
0
#
7
d
Age (weeks)
40
40 Age (weeks)
Fig. 4. Semiquantitative analysis of histological changes in the kidney. Glomerular adhesion (a), interstitial fibrosis (b), OPN (c), and number of ED1-positive cells (d). # p < 0.05; vs. control kidney (a, c, d), and non-IR kidney (b), + p < 0.05; vs. control kidney (c, d).
Table 2. Comparison of parameters in
7-week-old rats
7- and 40-week-old SD rats at 7 days after unilateral IR
sham Body weight, g BP, mm Hg Cre, mg/l
IR
40-week-old rats p
234.3 ± 5.9 284.3 ± 1.9
