Yonago Acta medica 2001;41:37–44

Effects of Testosterone Replacement on Renal Function and Apoptosis
on Mesangial and Renal Tubule Cells in Rats
Kuniyasu Muraoka
Department of Urology and First Department of Pathology, Tottori University Faculty of
Medicine, Yonago 683-0826 JapanThe effect of testosterone replacement in middle-aged rats on renal function andapoptosis of mesangial and renal tubule cells was evaluated. Middle-aged (13-monthold) male Wistar rats were divided into 2 groups, one of which was given high-dose andthe other low-dose testosterone replacement for 2 months. Experiments were performed
with the testosterone replacement groups and a non-replacement group when they hadreached the age of 15 months. Serum levels of total and free testosterone were measured,and renal function was evaluated by glomerular filtration rate (GFR), serum levels ofcreatinine, blood urea nitrogen (BUN), urinary protein and N-acetyl-β-D-glucosaminidase (NAG). Apoptosis was evaluated by counting the number of terminal deoxynucleotidyl transferase-mediated deUTP-biotin nick end labeling (TUNEL)-positive cells in
the mesangial and renal tubule cells. Correlations between total and free testosterone
and each parameter were evaluated. Also, 12-week-old rats were evaluated in the samemanner as the age control. Among 15-month-old rats, testosterone replacement did notaffect serum creatinine, BUN or urinary protein, but the GFR was found to correlate
negatively with the total and free testosterone level, and the urinary NAG level was
found to correlate positively with the free testosterone level to a significant extent (P <0.01). Percentages of apoptosis of mesangial and renal tubule cells were found tocorrelate positively with the total and free testosterone levels to a significant extent (P <0.01). In conclusion, the results of this study suggested that testosterone replacement
affected the deterioration of renal function in middle-aged rats.
Key words: apoptosis; renal function; testosterone

in renal function and in mesangial and renal
tubule cells when aging rats are subjected to testosterone replacement.
In the present study, the effects of testosterone replacement on renal function and on
apoptosis of mesangial cells and renal tubule
cells was evaluated in rats.

It has been reported that aging females and castrated male rats excrete less protein in the urine
than aging male rats (Gafter et al., 1990), and it
has been concluded that androgens are a risk
factor in the development of glomerulosclerotic
injury (Mulronery et al., 1999; Reckelhoff etal., 1997; Neugarten et al., 1996).
In cultured rat mesangial cells, mesangial
cells derived from aged rats show an increased
rate of apoptosis when compared to those fromyoung rats, and the rate of mesangial cell apoptosis increases as the concentration of testosterone is increased (Singhal et al., 1997).
It is not known what kind of changes occur

Materials and Methods
Male Wistar rats were obtained (Shimizu Experimental Material Company, Kyoto, Japan).
All rats were housed in a temperature-controlled

Abbreviations: BUN, blood urea nitrogen; dUTP, deoxyuridine triphosphate;GFR, glomerular filtration rate;
NAG, N-acetyl-β-D-glucosaminidase; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nickend labeling37

K. Muraokaroom (22 ± 1˚C) with 14 h of illumination daily(0600–2000) and given food and water. Experiments were performed when the rats had reached the age of 12 weeks (Group A, n = 10) and 15
months. The 15-month-old rats were divided
into 3 groups as in the following: rats with notestosterone replacement (Group B, n = 6), lowdose testosterone replacement (Group C, n = 8)
and high-dose testosterone replacement (GroupD, n = 8).
In the testosterone replacement groups(Groups C and D), rats were anesthetized forsurgery by inhalation of diethyl ether. A silastic
tube (Kaneka Medix Corp., Osaka, Japan) 3 cm
in length and containing 40 mg of testosterone
powder (Sigma, St. Louis, MO) was subcutaneously implanted in the backs of the rats. Foursilastic tubes (Group C) or 8 silastic tubes (GroupD) were implanted in rats 13 months old. Testosterone replacement was continued for 2
months.
In the experiment, 24-h urine collections
were performed for fasting rats which werehoused in individual metabolic cages, and urine
levels of creatinine, protein and N-acetyl-β-Dglucosaminidase (NAG) were evaluated. Aftercompletion of urine volume for each day, rats
were anesthetized with ether, and blood waswithdrawn into heparinized syringes by vena
cava puncture in order to determine the serum
levels of total and free testosterone, creatinine
and blood urea nitrogen (BUN), and then bilateral nephrectomy was performed, and therats were killed. Each kidney was cut free ofsurrounding tissue, weighed on a Mettler
Basbal scale (Delta Range, Tokyo, Japan), cut
by a coronal section through the mid-portion of
the kidney, and fixed in 10% buffered formalin.
Several paraffin blocks were prepared from thissection and several 3-µm sections were cut
from each paraffin block and stained via terminal deoxynucleotidyl transferase-mediateddUTP-biotin nick end labeling (TUNEL).
TUNEL staining was performed with an
ApopTag in situ apoptosis detection kit (Oncor,
Gaithersburg, MD). Semiquantitative analysis
was performed by counting TUNEL-positive
cells per field at × 400 magnification. The

Table 1. Serum concentration of testosterone
Total testosterone(ng/mL)Free testosterone(ng/mL)Group A
2.0 ± 0.4
8.3 ± 1.9
Group B
0.8 ± 0.1
4.2 ± 1.1
Group C3.7 ± 0.4
14.5 ± 3.6Group D
5.0 ± 1.1
20.0 ± 6.7
Data are expressed as mean ± SD.
Statistical analysis by Mann-Whitney U-test.
Group A versus Group B, P < 0.05.
Group B versus Group C, P < 0.05.
Group B versus Group D, P < 0.05.

mean number of TUNEL-positive cells in 50
randomly selected fields in each kidney wascalculated.

Urine level of creatinine, protein and NAG
Urinary creatinine was determined by the thealkaline picrate method, urinary protein wasdetermined by the pyrogallolred method, andurinary NAG was determined by colorimetry by
the Special Reference Laboratory (Hiroshima,
Japan) .

Serum levels of total and free testosterone, creatinine and BUN
Total and free testosterone levels were assessedby radioimmunoassay using a kit from Nihon
DPC Corporation (Tokyo, Japan). The BUN
level was assessed by the urease-UV method,
and the serum creatinine level was determinedby the alkaline picrate method.

Calculation of the glomerular filtration
rate (GFR)
The GFR was determined by the creatinine
clearance expressed per gram of kidney weight.

Statistical analysis
The data are presented as mean ± SD; KruskalWallis one-way analysis of variance was performed. When a difference found to be significant,38

Effects of testosterone replacement

Table 2. Effects of testosterone replacement on renal function
Serum creatinine(mg/dL)

BUN(mg/dL)Urinary protein(mg/dL)Urinary NAG
(U/L)

GFR
(mL/min/kw)

Group A
0.2 ± 0.1
15.6 ± 1.4
65.8 ± 27.3
5.9 ± 2.3
174.16 ± 37.90
Group B
0.3 ± 0.1
21.1 ± 1.6
327.2 ± 108.9
4.6 ± 7.3
124.40 ± 9.94
Group C
0.4 ± 0.1
20.4 ± 2.0
445.6 ± 181.4
14.0 ± 10.8
104.47 ± 8.22
Group D
0.3 ± 0.1
21.3 ± 2.3
899.1 ± 487.4
11.3 ± 11.3
98.51 ± 17.36
Data are expressed as mean ± SD.
BUN, blood urea nitrogen; GFR, glomerular filtration rate; kw, kidney weight (g); NAG, N-acetyl-β-Dglucosaminidase; NS, not significant.
Statistical analysis by Mann-Whitney U-test.
Group A versus Group B: serum creaninine, BUN and urine NAG = NS; urinary protein and GFR = P < 0.05.
Group B versus Group C: serum creaninine, BUN and urinary protein = NS; urine NAG and GFR = P < 0.05.
Group B versus Group D: serum creaninine and BUN = NS; urinary protein, urine NAG and GFR = P < 0.05.

the Mann-Whitney U-test for 2 independent samples was used to compare each of the 2 groups.
Statistical significance was defined as P < 0.05.

Apoptosis of mesangial and renal tubule
cells
TUNEL-positive mesangial and renal tubule
cells of high-dose testosterone replacement rats
(Group D) are shown in Figs. 1a and b. The
percentages of apoptotic mesangial and renal
tubule cells were similar to those of Groups A
and B (Table 3). Among groups of 15-monthold rats, mesangial and renal tubule cells ofGroups C and D showed greater percentages of
TUNEL-positive cells when compared to thoseof Group B.

Results

Serum concentration of testosterone
The serum levels of total and free testosteronein Group B were significantly lower than thosein Group A. Among groups of 15-month-old
rats, the serum levels of total and free testosterone in Groups C and D were significantly
higher than those in Group B (Table 1).

Serum creatinin, BUN, urinary protein,urine NAG and GFRCorrelations between urinary protein,urinary NAG, GFR, apoptosis of mesangial and renal tubule cells and total andfree testosterone

The serum creatinin, BUN and urine NAG
levels of Groups A and B were similar to that of
the control group. The level of urinary protein
in Group B was significantly higher than that ofGroup A, and GFR in Group B was significantly lower than that in Group A.
Among groups of 15-month-old rats, testosterone replacement groups (Groups C and D)
showed significantly higher levels of urinary
protein and NAG, significantly lower values of
GFR, but no significant difference in serum
creatinin and BUN (Table 2).

In the 15-month-old rats, the correlation between the serum concentration of total and freetestosterone and each parameter was examinedusing linear regression analysis. The urinary
protein level was not found to correlate with thetotal and free testosterone levels (Figs. 2a andb). The urinary NAG level was found to correlate positively with the free testosterone levelbut not with the total testosterone levels (Figs.
3a and b). The GFR was found to correlate
negatively with the total and free testosterone
levels (Figs. 4a and b). Percentages of apopto-

39

K. MuraokaabFig. 1. TUNEL-positive cells of a high-dose testosterone replacement rat at the age of 15 months.
a: TUNEL-positive mesangial cells (original magnification × 400).
b: TUNEL-positive renal tubule cells (original magnification × 400).

sis of mesangial and renal tubule cells were
found to correlate positively with the total andfree testosterone levels (Figs. 5a, 5b, 6a and 6b).

Discussion
In a previous study involving male Wistar rats,
the GFR began diminishing at 16 months(Tanaka et al., 1995). In this previous study, 2months of testosterone replacement at 1340

Effects of testosterone replacement

Table 3. Effects of testosterone replacement
on apoptosis of mesangial cells and renal
tubulesmonths old accelerated a reduction of the GFR.
The direct effects of testosterone on cellularcollagen synthesis have not been extensively
studied; however, most available data suggeststhat it has a stimulatory effect (Fischer et al.,
1985; Franchimont et al., 1991). For example,
testosterone increases collagen synthesis by
vascular smooth muscle cells in culture(Leitman et al., 1994), and administration of
testosterone increases the accumulation ofcollagen and elastin in the aorta of normal andcholesterol-fed animals (Silbiger et al., 1995).
It has been suggested that some changes related
to collagen may occur along with a significant
reduction in the GFR in rats that have undergone testosterone replacement.
Rats that have undergone testosterone replacement exhibited proteinuria. In this study,
urine albumin was not detected, and it is nota% Apoptotic
mesangial cells

Group A
0.96 ± 0.25
0.46 ± 0.21
Group B
1.07 ± 0.36
0.87 ± 0.27
Group C
1.47 ± 0.40
1.23 ± 0.24
Group D
2.05 ± 0.60
1.60 ± 0.35
Data are expressed as mean ± SD.
Statistical analysis by Mann-Whitney U-test.
Group A versus Group B, not significant.
Group B versus Group C, P < 0.05.
Group B versus Group D, P < 0.05.

clear what kind of protein was excreted, but it
might be that proteinuria evolved from a leak inthe basement membrane. Excretion of sexdependent α2u-globulin (Roy et al., 1966;

y = 275.537 + 89.501 x
r = 0.421
P = 0.510(mg/dL)1800by = 360.238 + 15.900 x
r = 0.313
P = 0.156(mg/dL)
1800
1600

Urinary protein

1600

Urinary protein% Apoptotic
renal tubule cells

1400
1200
1000
800
600
400

1400
1200
1000
800
600
400
200

200

0

0
0

1

2

3

4

5

6

7

8

(mg/dL)

0

5

Total testosterone

10

15

20

25

30

35

40 (pg/mL)

Free testosterone

Fig. 2. Correlation between urinary protein and total and free testosterone in 15-month-old rats.

aby = 2.577 + 2.331 x
r = 0.416
P = 0.543

(U/L)
35

35
30

Urinary NAG

30Urinary NAG

y = 2.051 + 0.631 x
r = 0.458
P < 0.05

(U/L)

25
20
15
10

25
20
15
10
5

5

0

0
0

1

2

3

4

5

6

7

8 (mg/dL)

Total testosterone

0

5

10

15

20

25

30

35

40 (pg/mL)

Free testosterone

Fig. 3. Correlation between urinary NAG and total and free testosterone in 15-month-old rats. NAG, Nacetyl-β-D-glucosaminidase.

41

K. Muraoka

a
130

130

120

120

110
100

y = 130.296 – 1.641 x
r = 0.792
P < 0.01(mL/min/kw)
140

GFR

GFR

b

y = 130.371 – 6.693 x
r = 0.770
P < 0.01(mL/min/kw)
140

110
100

90

90

80

80

70

70
0

1

2

3

4

5

6

7

8

(mg/dL)

0

5

10

Total testosterone

15

20

25

30

35

40 (pg/mL)

Free testosterone

Fig. 4. Correlation between GFR and total and free testosterone in 15-month-old rats. GFR, glomerular
filtration rate; kw, kidney weight (g).

Neuhaus et al., 1975) can be abolished by castration of male rats (Irwin et al., 1971). It would
seem that sex-dependent globulin could only
slightly contribute to proteinuria. It was foundthat urinary NAG increased and correlated
positively with the free testosterone level but
not with the total testosterone level. NAG is
one of the glycoproteinases in lysosome, andproximal renal tubule epithelial cells contain alot of NAG. Urinary NAG increases due to an
injured proximal tubule. It is believed that freetestosterone may injure proximal tubules.
In addition, it was demonstrated that testosterone replacement increased the number of
TUNEL-positive cells in the mesangial andrenal tubule cells when compared to the control
group in middle-aged rats. Cultured mesangial

cell apoptosis in male rats increases with thelevel of testosterone (Singhal et al., 1997). The
present study demonstrated that this phenomenon occurred similarly in vivo. In addition,
renal tubule cell apoptosis in male rats also increased with the level of testosterone. It is
known that cultured mesangial cells from malerats contain nuclear receptors for testosterone(Neugarten et al., 1994). In addition, it is believed that renal tubule cells contain nuclear receptors for testosterone (Davidoff et al., 1980).
Free testosterone has true androgen activity, so
it is believed that some changes in mesangial
and renal tubule cells may occur with a significant increase in the number of TUNELpositive cells by the direct effect of free testosterone.

aby = 0.733 + 0.266 x
r = 0.801
P < 0.01% Apoptotic
mesangial cells

3.5
3.0

3.5

% Apoptotic
mesangial cells

(%)

2.5
2.0
1.5
1.0
5.0
0

y = 0.674 + 0.070 x
r = 0.882
P < 0.01

(%)

3.0
2.5
2.0
1.5
1.0
5.0
0

0

1

2

3

4

5

6

7

8

(mg/dL)

Total testosterone

0

5

10

15

20

25

30

35

40 (pg/mL)

Free testosterone

Fig. 5. Correlation between % apoptosis of mesangial cells and total and free testosterone in 15-month-old
rats.

42

Effects of testosterone replacement

a

b

2.4

y = 0.651 + 0.470 x
r = 0.903
P < 0.01(%)

y = 0.646 + 0.183 x
r = 0.832
P < 0.01% Apoptotic
renal tubule cells% Apoptotic
renal tubule cells

(%)

2.0
1.6
1.2
0.8
0.4

2.4
2.0
1.6
1.2
0.8
0.4

0

1

2

3

4

5

6

7

8 (mg/dL)

Total testosterone

0

5

10

15

20

25

30

35

40 (pg/mL)

Free testosterone

Fig. 6. Correlation between % apoptosis of renal tubule cells and total and free testosterone in 15-month-old
rats.

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3 Fischer GM, Bashey RI, Rosenbaum H, Lyttle
CR. A possible mechanism in arterial wall for
mediation of sex difference in atherosclerosis.
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On the other hand, it was surmised that testosterone replacement caused a decrease of gonadotrophic hormone by a feedback mechanism. However, the relation between the gonadotrophic hormone and mesangial and renal
tubule cells is not clear. Further, multiple apoptosis signaling pathways have been described,
but further studies should be performed to determine the apoptosis pathway for mesangial
and renal tubule cells treated with testosterone.
The present study is the first report showingthat testosterone replacement in middle-aged
rats increased the rate of apoptosis of mesangial
and renal tubule cells and the urine level of
NAG and decreased the GFR. The concept that
androgens are a risk factor for renal dysfunction
is supported by the current findings, but further
studies are needed to clarify their association
with testosterone.

Acknowledgments: The author would like to thankProfessor Ikuo Miyagawa of the Department ofUrology, Tottori University Faculty of Medicine, for
his kind advice and valuable suggestions concerning
this study.

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Received December 15, 2000; accepted December 25, 2000
Corresponding author: Dr. Kuniyasu Muraoka

44