252 HormoIL Metab. Res. 9 (1977) 252
© Georg Thieme Verlag Stuttgart Testosterone Metabolism of Isolated Human Kidney Tubules
H. Bojar, C. Funcke, R. Dreyfürst, U. Matthiesen and W. Staib Inst. für Physiol. ehern. 11, Universität Düsseldorf, Germany
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Human kidney tissue used in this study was obtained from 7 male patients, aged 49 to 79, who were nephrectomized because of renal cell carcinoma. In all cases, the carcinoma was located only at the upper pole of the kidney. Specimens were taken from those parts of the organ which by histological examination proved to be free of infIltrating cancer tissue. Kidney tubules and cells were isoiated by the method of BojQr et QL (1975). Driefly, slices of human kidney were incubated at 37 0C in Krebs-Ringer bicarbonate (KRD) buffer (pH 7.4), containing crude coUagenase (10 mgJg wet weight of slices). After 20 min of incubation ea02 (final concentration 5 mM) was added and incubation continued for another 50 miIL Subsequently, unbroken tissue fragments were allowed to sediment (20 seconds). A mixed suspension of isolated kidney tubules and cells was obtained by centrüugation of the supernatant at 50 x g for 1 miIL 111e usual yield amounted to 80 % of the wet weisht of slices. 111e isoiated tubules and cells were viable as judged by nuclear trypan blue exclusion (> 90 %) as weU as their ability to form glucose from different substrates at 10 mM (fructose: 11.8 ± 0.45 ",moles glucose/g wet weight x hr, lactate: 5.2 :t 0.19 ",moles/g wet weight x hr, pyruvate: 3.9 ± 0.21 ",moles/g wet weisht x hr; mean :tSEM, n = 5). As further evidence of their metabolic integrity, the degradation of testosterone was investisated. Isolated tubules and ceUs (25.3 :t 1.6 Mg of protein per individual reaction; mean :t SEM, n = 7), suspended in 2 mI of KRD buffer, containing 5.5 mM glucose, were preincubated in commercially available polyethylen vials (ps phase: 95% 02, 5 % C02) undel constant shaking (120 oscillatiolll/min) for 5 min at 37 0c. Subsequently, 300 ",g of unlabeUed testosterone and 0.15 folg of 4- 14 C-testosterone (specific activity 59.4 mCi/mMol), dissolved in 20 ",1 CH30H, were added. After 75 min of incubation, the free steroid metabolites were extracted with ether. In the organic phase 88.6 :t 1.5 % (mean :t SE~.i, n = 7) of the total radioactivity was detected. 111e aqueous phase was subjected to hydroxide b, of hyamine (3 hr at 60 OC) and found to contain 5.0 :t 0.44% (mean :t SEM) of the 14C activity. 111us, approximately 93.6% of the initial!y added hormone could be recovered. 111e decrease of the total radioactivity in the organic phase and the c) Start uniden- testoste- 4-androsten-3,17-diincrease in 14C activity in the aqueous phase were found to tified rone one; 19.2 ± 8", be statistically sisnificant (organic phase: p < 0.02, aqueous 246.4:t (mean ± SEM, phase: p < 0.0001). 111e pattern of conjugates in the aqueous n = 6) 4.8 118 phase is under current investigation. The separation of the (mean ± metabolites of the organic phase was achieved by one-dimenSEM, n =6) sional development on TLC-plates (Silica Gel 60 F 254 ) in Fis. 1. Pattern of unconjugated testosterone metabolites sesystem 1 (chloroforme/acetone 92.5/7.5) foUowed by system parated by TLC. 2 (cyc1ohexane/toluene/ethylacetate/methanol 25/25/50/12). X-ray films (Osray T-4, Agfa-Gevaert) were exposed to the TLC-plates for 9 days. A representative TLC-radiochromatogram (Fig. la) and the corresponding X-ray film (Fig. Ib) of the organic phase obtained after 75 min of incubation are shown. 111e radioactive zones were then eluted with CH30H, quantified by LSC and identified as testosterone and 4-androsten-3,17-dione (Fis. lc) by combined gas chromatography/mass spectrometry (Varian Mat CH7, Varian aerograph series 1700, 3% OVI on Chromosorb W-HP AW DMCS at 250 oc, Bieman-Watson Helium separator, ion source 160°C, 70 eV, He 50 mI/min). 111e decrease in the concentration of testosterone (p < 0.05) and the formation of 4-androsten-3,17-dione (p < 0.01) were calcuIated to be statistically sisnificant. In contrast, in contro! experiments with heat inactivated (100 oc, 15 min) kidney tubules essentially no testosterone metabolites could be detected. It appears that human kidney is enzymatica1ly equipped to playapart in the extrahepatic metabolism of testosterone, and that enzymatically iso!ated human kidney tubules and cells maintain theil capacity to degrade the male sex hormone.
.,
o
GO
00
Reference: Bojar, H., K. BQlzer, F. BoeminghQus, W. Staib: Z. Klin. Chem. K1in. Biochem. 13: 31-35 (1975). -
Requests for reprints should be addressed to: H. Bojar, Dept. PhysioL Chem. 11, Univ. Düsseldorf, 4000 Diisseldorf, Moorenstraße (Germany).
Rec.:
m June
1976
Acc.: 21 Feb. 1977
© Georg Thieme Verlag Stuttgart Testosterone Metabolism of Isolated Human Kidney Tubules
H. Bojar, C. Funcke, R. Dreyfürst, U. Matthiesen and W. Staib Inst. für Physiol. ehern. 11, Universität Düsseldorf, Germany
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Human kidney tissue used in this study was obtained from 7 male patients, aged 49 to 79, who were nephrectomized because of renal cell carcinoma. In all cases, the carcinoma was located only at the upper pole of the kidney. Specimens were taken from those parts of the organ which by histological examination proved to be free of infIltrating cancer tissue. Kidney tubules and cells were isoiated by the method of BojQr et QL (1975). Driefly, slices of human kidney were incubated at 37 0C in Krebs-Ringer bicarbonate (KRD) buffer (pH 7.4), containing crude coUagenase (10 mgJg wet weight of slices). After 20 min of incubation ea02 (final concentration 5 mM) was added and incubation continued for another 50 miIL Subsequently, unbroken tissue fragments were allowed to sediment (20 seconds). A mixed suspension of isolated kidney tubules and cells was obtained by centrüugation of the supernatant at 50 x g for 1 miIL 111e usual yield amounted to 80 % of the wet weisht of slices. 111e isoiated tubules and cells were viable as judged by nuclear trypan blue exclusion (> 90 %) as weU as their ability to form glucose from different substrates at 10 mM (fructose: 11.8 ± 0.45 ",moles glucose/g wet weight x hr, lactate: 5.2 :t 0.19 ",moles/g wet weight x hr, pyruvate: 3.9 ± 0.21 ",moles/g wet weisht x hr; mean :tSEM, n = 5). As further evidence of their metabolic integrity, the degradation of testosterone was investisated. Isolated tubules and ceUs (25.3 :t 1.6 Mg of protein per individual reaction; mean :t SEM, n = 7), suspended in 2 mI of KRD buffer, containing 5.5 mM glucose, were preincubated in commercially available polyethylen vials (ps phase: 95% 02, 5 % C02) undel constant shaking (120 oscillatiolll/min) for 5 min at 37 0c. Subsequently, 300 ",g of unlabeUed testosterone and 0.15 folg of 4- 14 C-testosterone (specific activity 59.4 mCi/mMol), dissolved in 20 ",1 CH30H, were added. After 75 min of incubation, the free steroid metabolites were extracted with ether. In the organic phase 88.6 :t 1.5 % (mean :t SE~.i, n = 7) of the total radioactivity was detected. 111e aqueous phase was subjected to hydroxide b, of hyamine (3 hr at 60 OC) and found to contain 5.0 :t 0.44% (mean :t SEM) of the 14C activity. 111us, approximately 93.6% of the initial!y added hormone could be recovered. 111e decrease of the total radioactivity in the organic phase and the c) Start uniden- testoste- 4-androsten-3,17-diincrease in 14C activity in the aqueous phase were found to tified rone one; 19.2 ± 8", be statistically sisnificant (organic phase: p < 0.02, aqueous 246.4:t (mean ± SEM, phase: p < 0.0001). 111e pattern of conjugates in the aqueous n = 6) 4.8 118 phase is under current investigation. The separation of the (mean ± metabolites of the organic phase was achieved by one-dimenSEM, n =6) sional development on TLC-plates (Silica Gel 60 F 254 ) in Fis. 1. Pattern of unconjugated testosterone metabolites sesystem 1 (chloroforme/acetone 92.5/7.5) foUowed by system parated by TLC. 2 (cyc1ohexane/toluene/ethylacetate/methanol 25/25/50/12). X-ray films (Osray T-4, Agfa-Gevaert) were exposed to the TLC-plates for 9 days. A representative TLC-radiochromatogram (Fig. la) and the corresponding X-ray film (Fig. Ib) of the organic phase obtained after 75 min of incubation are shown. 111e radioactive zones were then eluted with CH30H, quantified by LSC and identified as testosterone and 4-androsten-3,17-dione (Fis. lc) by combined gas chromatography/mass spectrometry (Varian Mat CH7, Varian aerograph series 1700, 3% OVI on Chromosorb W-HP AW DMCS at 250 oc, Bieman-Watson Helium separator, ion source 160°C, 70 eV, He 50 mI/min). 111e decrease in the concentration of testosterone (p < 0.05) and the formation of 4-androsten-3,17-dione (p < 0.01) were calcuIated to be statistically sisnificant. In contrast, in contro! experiments with heat inactivated (100 oc, 15 min) kidney tubules essentially no testosterone metabolites could be detected. It appears that human kidney is enzymatica1ly equipped to playapart in the extrahepatic metabolism of testosterone, and that enzymatically iso!ated human kidney tubules and cells maintain theil capacity to degrade the male sex hormone.
.,
o
GO
00
Reference: Bojar, H., K. BQlzer, F. BoeminghQus, W. Staib: Z. Klin. Chem. K1in. Biochem. 13: 31-35 (1975). -
Requests for reprints should be addressed to: H. Bojar, Dept. PhysioL Chem. 11, Univ. Düsseldorf, 4000 Diisseldorf, Moorenstraße (Germany).
Rec.:
m June
1976
Acc.: 21 Feb. 1977
