Effects of Testosterone Replacement on Renal Function and Apoptosis on Mesangial and Renal Tubule Cells in Rats

Transcription

1 Yongo Act medic 21;41:37 44 Effects of Testosterone Replcement on Renl Function nd Apoptosis on Mesngil nd Renl Tuule Cells in Rts Kuniysu Murok Deprtment of Urology nd First Deprtment of Pthology, Tottori University Fculty of Medicine, Yongo Jpn The effect of testosterone replcement in middle-ged rts on renl function nd poptosis of mesngil nd renl tuule cells ws evluted. Middle-ged (13-monthold) mle Wistr rts were divided into 2 groups, one of which ws given high-dose nd the other low-dose testosterone replcement for 2 months. Experiments were performed with the testosterone replcement groups nd non-replcement group when they hd reched the ge of 15 months. Serum levels of totl nd free testosterone were mesured, nd renl function ws evluted y glomerulr filtrtion rte (GFR), serum levels of cretinine, lood ure nitrogen (BUN), urinry protein nd N-cetyl-β-D-glucosminidse (NAG). Apoptosis ws evluted y counting the numer of terminl deoxynucleotidyl trnsferse-medited deutp-iotin nick end leling (TUNEL)-positive cells in the mesngil nd renl tuule cells. Correltions etween totl nd free testosterone nd ech prmeter were evluted. Also, 12-week-old rts were evluted in the sme mnner s the ge control. Among 15-month-old rts, testosterone replcement did not ffect serum cretinine, BUN or urinry protein, ut the GFR ws found to correlte negtively with the totl nd free testosterone level, nd the urinry NAG level ws found to correlte positively with the free testosterone level to significnt extent (P <.1). Percentges of poptosis of mesngil nd renl tuule cells were found to correlte positively with the totl nd free testosterone levels to significnt extent (P <.1). In conclusion, the results of this study suggested tht testosterone replcement ffected the deteriortion of renl function in middle-ged rts. Key words: poptosis; renl function; testosterone It hs een reported tht ging femles nd cstrted mle rts excrete less protein in the urine thn ging mle rts (Gfter et l., 199), nd it hs een concluded tht ndrogens re risk fctor in the development of glomerulosclerotic injury (Mulronery et l., 1999; Reckelhoff et l., 1997; Neugrten et l., 1996). In cultured rt mesngil cells, mesngil cells derived from ged rts show n incresed rte of poptosis when compred to those from young rts, nd the rte of mesngil cell poptosis increses s the concentrtion of testosterone is incresed (Singhl et l., 1997). It is not known wht kind of chnges occur Arevitions: BUN, lood ure nitrogen; dutp, deoxyuridine triphosphte;gfr, glomerulr filtrtion rte; NAG, N-cetyl-β-D-glucosminidse; TUNEL, terminl deoxynucleotidyl trnsferse-medited dutp-iotin nick end leling 37 in renl function nd in mesngil nd renl tuule cells when ging rts re sujected to testosterone replcement. In the present study, the effects of testosterone replcement on renl function nd on poptosis of mesngil cells nd renl tuule cells ws evluted in rts. Mterils nd Methods Mle Wistr rts were otined (Shimizu Experimentl Mteril Compny, Kyoto, Jpn). All rts were housed in temperture-controlled

2 K. Murok room (22 ± 1 C) with 14 h of illumintion dily (6 2) nd given food nd wter. Experiments were performed when the rts hd reched the ge of 12 weeks (Group A, n = 1) nd 15 months. The 15-month-old rts were divided into 3 groups s in the following: rts with no testosterone replcement (Group B, n = 6), lowdose testosterone replcement (Group C, n = 8) nd high-dose testosterone replcement (Group D, n = 8). In the testosterone replcement groups (Groups C nd D), rts were nesthetized for surgery y inhltion of diethyl ether. A silstic tue (Knek Medix Corp., Osk, Jpn) 3 cm in length nd contining 4 mg of testosterone powder (Sigm, St. Louis, MO) ws sucutneously implnted in the cks of the rts. Four silstic tues (Group C) or 8 silstic tues (Group D) were implnted in rts 13 months old. Testosterone replcement ws continued for 2 months. In the experiment, 24-h urine collections were performed for fsting rts which were housed in individul metolic cges, nd urine levels of cretinine, protein nd N-cetyl-β-Dglucosminidse (NAG) were evluted. After completion of urine volume for ech dy, rts were nesthetized with ether, nd lood ws withdrwn into heprinized syringes y ven cv puncture in order to determine the serum levels of totl nd free testosterone, cretinine nd lood ure nitrogen (BUN), nd then ilterl nephrectomy ws performed, nd the rts were killed. Ech kidney ws cut free of surrounding tissue, weighed on Mettler Bsl scle (Delt Rnge, Tokyo, Jpn), cut y coronl section through the mid-portion of the kidney, nd fixed in 1% uffered formlin. Severl prffin locks were prepred from this section nd severl 3-µm sections were cut from ech prffin lock nd stined vi terminl deoxynucleotidyl trnsferse-medited dutp-iotin nick end leling (TUNEL). TUNEL stining ws performed with n ApopTg in situ poptosis detection kit (Oncor, Githersurg, MD). Semiquntittive nlysis ws performed y counting TUNEL-positive cells per field t 4 mgnifiction. The Tle 1. Serum concentrtion of testosterone Totl testosterone (ng/ml) (ng/ml) Group A 2. ± ± 1.9 Group B.8 ± ± 1.1 Group C 3.7 ± ± 3.6 Group D 5. ± ± 6.7 Dt re expressed s men ± SD. Sttisticl nlysis y Mnn-Whitney U-test. Group A versus Group B, P <.5. Group B versus Group C, P <.5. Group B versus Group D, P <.5. men numer of TUNEL-positive cells in 5 rndomly selected fields in ech kidney ws clculted. Urine level of cretinine, protein nd NAG Urinry cretinine ws determined y the the lkline picrte method, urinry protein ws determined y the pyrogllolred method, nd urinry NAG ws determined y colorimetry y the Specil Reference Lortory (Hiroshim, Jpn). Serum levels of totl nd free testosterone, cretinine nd BUN Totl nd free testosterone levels were ssessed y rdioimmunossy using kit from Nihon DPC Corportion (Tokyo, Jpn). The BUN level ws ssessed y the urese-uv method, nd the serum cretinine level ws determined y the lkline picrte method. Clcultion of the glomerulr filtrtion rte (GFR) The GFR ws determined y the cretinine clernce expressed per grm of kidney weight. Sttisticl nlysis The dt re presented s men ± SD; Kruskl- Wllis one-wy nlysis of vrince ws performed. When difference found to e significnt, 38

3 Effects of testosterone replcement Tle 2. Effects of testosterone replcement on renl function Serum cretinine BUN Urinry protein Urinry NAG GFR (mg/dl) (mg/dl) (mg/dl) (U/L) (ml/min/kw) Group A.2 ± ± ± ± ± 37.9 Group B.3 ± ± ± ± ± 9.94 Group C.4 ± ± ± ± ± 8.22 Group D.3 ± ± ± ± ± Dt re expressed s men ± SD. BUN, lood ure nitrogen; GFR, glomerulr filtrtion rte; kw, kidney weight (g); NAG, N-cetyl-β-Dglucosminidse; NS, not significnt. Sttisticl nlysis y Mnn-Whitney U-test. Group A versus Group B: serum creninine, BUN nd urine NAG = NS; urinry protein nd GFR = P <.5. Group B versus Group C: serum creninine, BUN nd urinry protein = NS; urine NAG nd GFR = P <.5. Group B versus Group D: serum creninine nd BUN = NS; urinry protein, urine NAG nd GFR = P <.5. the Mnn-Whitney U-test for 2 independent smples ws used to compre ech of the 2 groups. Sttisticl significnce ws defined s P <.5. Results Serum concentrtion of testosterone The serum levels of totl nd free testosterone in Group B were significntly lower thn those in Group A. Among groups of 15-month-old rts, the serum levels of totl nd free testosterone in Groups C nd D were significntly higher thn those in Group B (Tle 1). Serum cretinin, BUN, urinry protein, urine NAG nd GFR The serum cretinin, BUN nd urine NAG levels of Groups A nd B were similr to tht of the control group. The level of urinry protein in Group B ws significntly higher thn tht of Group A, nd GFR in Group B ws significntly lower thn tht in Group A. Among groups of 15-month-old rts, testosterone replcement groups (Groups C nd D) showed significntly higher levels of urinry protein nd NAG, significntly lower vlues of GFR, ut no significnt difference in serum cretinin nd BUN (Tle 2). Apoptosis of mesngil nd renl tuule cells TUNEL-positive mesngil nd renl tuule cells of high-dose testosterone replcement rts (Group D) re shown in Figs. 1 nd. The percentges of poptotic mesngil nd renl tuule cells were similr to those of Groups A nd B (Tle 3). Among groups of 15-monthold rts, mesngil nd renl tuule cells of Groups C nd D showed greter percentges of TUNEL-positive cells when compred to those of Group B. Correltions etween urinry protein, urinry NAG, GFR, poptosis of mesngil nd renl tuule cells nd totl nd free testosterone In the 15-month-old rts, the correltion etween the serum concentrtion of totl nd free testosterone nd ech prmeter ws exmined using liner regression nlysis. The urinry protein level ws not found to correlte with the totl nd free testosterone levels (Figs. 2 nd ). The urinry NAG level ws found to correlte positively with the free testosterone level ut not with the totl testosterone levels (Figs. 3 nd ). The GFR ws found to correlte negtively with the totl nd free testosterone levels (Figs. 4 nd ). Percentges of popto- 39

4 K. Murok Fig. 1. TUNEL-positive cells of high-dose testosterone replcement rt t the ge of 15 months. : TUNEL-positive mesngil cells (originl mgnifiction 4). : TUNEL-positive renl tuule cells (originl mgnifiction 4). sis of mesngil nd renl tuule cells were found to correlte positively with the totl nd free testosterone levels (Figs. 5, 5, 6 nd 6). Discussion In previous study involving mle Wistr rts, the GFR egn diminishing t 16 months (Tnk et l., 1995). In this previous study, 2 months of testosterone replcement t 13 4

5 Effects of testosterone replcement months old ccelerted reduction of the GFR. The direct effects of testosterone on cellulr collgen synthesis hve not een extensively studied; however, most ville dt suggests tht it hs stimultory effect (Fischer et l., 1985; Frnchimont et l., 1991). For exmple, testosterone increses collgen synthesis y vsculr smooth muscle cells in culture (Leitmn et l., 1994), nd dministrtion of testosterone increses the ccumultion of collgen nd elstin in the ort of norml nd cholesterol-fed nimls (Siliger et l., 1995). It hs een suggested tht some chnges relted to collgen my occur long with significnt reduction in the GFR in rts tht hve undergone testosterone replcement. Rts tht hve undergone testosterone replcement exhiited proteinuri. In this study, urine lumin ws not detected, nd it is not Tle 3. Effects of testosterone replcement on poptosis of mesngil cells nd renl tuules % Apoptotic % Apoptotic mesngil cells renl tuule cells Group A.96 ± ±.21 Group B 1.7 ± ±.27 Group C 1.47 ± ±.24 Group D 2.5 ± ±.35 Dt re expressed s men ± SD. Sttisticl nlysis y Mnn-Whitney U-test. Group A versus Group B, not significnt. Group B versus Group C, P <.5. Group B versus Group D, P <.5. cler wht kind of protein ws excreted, ut it might e tht proteinuri evolved from lek in the sement memrne. Excretion of sexdependent α2u-gloulin (Roy et l., 1966; Urinry protein (mg/dl) (mg/dl) Totl testosterone y = x r =.421 P =.51 y = x (mg/dl) r = P = (pg/ml) Fig. 2. Correltion etween urinry protein nd totl nd free testosterone in 15-month-old rts. Urinry protein Urinry NAG (U/L) y = x r =.416 P =.543 Urinry NAG (mg/dl) (U/L) y = x r =.458 P < (pg/ml) Totl testosterone Fig. 3. Correltion etween urinry NAG nd totl nd free testosterone in 15-month-old rts. NAG, N- cetyl-β-d-glucosminidse. 41

6 K. Murok GFR y = x (ml/min/kw) r = P < (mg/dl) Totl testosterone GFR y = x (ml/min/kw) r = P < (pg/ml) Fig. 4. Correltion etween GFR nd totl nd free testosterone in 15-month-old rts. GFR, glomerulr filtrtion rte; kw, kidney weight (g). Neuhus et l., 1975) cn e olished y cstrtion of mle rts (Irwin et l., 1971). It would seem tht sex-dependent gloulin could only slightly contriute to proteinuri. It ws found tht urinry NAG incresed nd correlted positively with the free testosterone level ut not with the totl testosterone level. NAG is one of the glycoproteinses in lysosome, nd proximl renl tuule epithelil cells contin lot of NAG. Urinry NAG increses due to n injured proximl tuule. It is elieved tht free testosterone my injure proximl tuules. In ddition, it ws demonstrted tht testosterone replcement incresed the numer of TUNEL-positive cells in the mesngil nd renl tuule cells when compred to the control group in middle-ged rts. Cultured mesngil cell poptosis in mle rts increses with the level of testosterone (Singhl et l., 1997). The present study demonstrted tht this phenomenon occurred similrly in vivo. In ddition, renl tuule cell poptosis in mle rts lso incresed with the level of testosterone. It is known tht cultured mesngil cells from mle rts contin nucler receptors for testosterone (Neugrten et l., 1994). In ddition, it is elieved tht renl tuule cells contin nucler receptors for testosterone (Dvidoff et l., 198). hs true ndrogen ctivity, so it is elieved tht some chnges in mesngil nd renl tuule cells my occur with significnt increse in the numer of TUNELpositive cells y the direct effect of free testosterone. % Apoptotic mesngil cells (%) y = x r =.81 P < (mg/dl) Totl testosterone % Apoptotic mesngil cells Fig. 5. Correltion etween % poptosis of mesngil cells nd totl nd free testosterone in 15-month-old rts. 42 y = x (%) 3.5 r =.882 P < (pg/ml)

7 Effects of testosterone replcement % Apoptotic renl tuule cells (%) y = x r =.832 P <.1 % Apoptotic renl tuule cells (%) y = x r =.93 P < (mg/dl) Totl testosterone (pg/ml) Fig. 6. Correltion etween % poptosis of renl tuule cells nd totl nd free testosterone in 15-month-old rts. On the other hnd, it ws surmised tht testosterone replcement cused decrese of gondotrophic hormone y feedck mechnism. However, the reltion etween the gondotrophic hormone nd mesngil nd renl tuule cells is not cler. Further, multiple poptosis signling pthwys hve een descried, ut further studies should e performed to determine the poptosis pthwy for mesngil nd renl tuule cells treted with testosterone. The present study is the first report showing tht testosterone replcement in middle-ged rts incresed the rte of poptosis of mesngil nd renl tuule cells nd the urine level of NAG nd decresed the GFR. The concept tht ndrogens re risk fctor for renl dysfunction is supported y the current findings, ut further studies re needed to clrify their ssocition with testosterone. Acknowledgments: The uthor would like to thnk Professor Ikuo Miygw of the Deprtment of Urology, Tottori University Fculty of Medicine, for his kind dvice nd vlule suggestions concerning this study. References 1 Dvidoff M, Cffier H, Schieler TH. Steroid hormone inding receptors in the rt kidney. Histochemistry 198;69: Frnchimont P, Bssleer C. Effects of hormones nd locl growth fctors on rticulr chondrocyte metolism. J Rheumtol 1991;18: Fischer GM, Bshey RI, Rosenum H, Lyttle CR. A possile mechnism in rteril wll for medition of sex difference in therosclerosis. Exp Mol Pthol 1985;43: Gfter U, Ben Bsst M, Levi J. Cstrtion inhiits glomerulr hypertrophy nd proteinuri in uninephrectomized mle rts. Eur J Clin Invest 199;4: Irwin JF, Lne SE, Neuhus OS. Synergistic effect of glucocorticoids nd ndrogens on the iosynthesis of sex-dependent protein in the mle rt. Biochim Biophys Act 1971;252: Leitmn DC, Benson SC, Johnson LK. Glucocorticoids stimulte collgen nd noncollgen protein synthesis in cultured vsculr smooth muscle cells. J Cell Biol 1994;98: Mulroney SE, Wod C, Johnson M, Pesce C. Gender differences in renl growth nd function fter uninephrectomy in dult rts. Kidney Int 1999;56: Neugrten J, Siliger S. Nucler receptor for estrdiol nd testosterone in mesngil cells. J Am Soc Nephrol 1994;5: Neugrten J, Siliger SR. Effects of sex hormones on mesngil cells. Am J Kidney Dis 1995;26: Neuhus OW, Flory W. The effect of dietry protein on the excretion of lph2u, the sexdependent protein of the dult mle rt. Biochim Biophys Act 1975;411: Reckelhoff JF, Zhng H, Grnger JP. Decline in renl hemodynmic function in ging SHR : role of ndrogens. Hypertension 1997;3(3 Pt 2): Roy AK, Neuhus OW. Identifiction of rt urinry proteins y zone nd immunoelectrophoresis. Proc Soc Exp Biol Med 1966;121: Siliger S, Neugrten J. The impct of gender on progressive renl functionl impirment. Am J 43

8 K. Murok Kidney Dis 1995;25: Singhl PC, Reddy K, Frnki N, Snwl V, Kpsi A, Gions N, et l. Age nd sex modulte renl expression SGP-2 nd trnsglutminse nd poptosis of splenocytes, thymocytes, nd mcrophge. J Invest Med 1997;45: Tnk A, Kyoukuw M, Mori T, Kwshim S. Accelertion of renl dysfunction with ging y the use of ndrogen in Wistr/Tw rts. In-vivo 1995;9: Received Decemer 15, 2; ccepted Decemer 25, 2 Corresponding uthor: Dr. Kuniysu Murok 44