Pft/igers Arch (1989) 4t4:634-639 N 'j fin Journal of Physiology Springet~Verlag1989 Study on the renal handling of sex dependent proteins in male rats studied by micropuncture techniques and by the isolated perfused rat kidney E. Jonas, J. M. Alt, H. J. Schurek, R. Brunkhorst, and H. Stolte Division of Nephrology, Department of Internal Medicine, Medical School Hannover, D-3000 Hannover, Federal Republic of Germany Abstract. The separation of sex dependent urinary proteins of the rat (SDP) by micro-disc electrophoresis results in at least eight well defined protein bands with differing molec- ular weights. The hepatic origin of a sex dependent urinary protein, named e2u-globulin, has been demonstrated before by other authors applying immunological methods. In the present study, it could been shown that SDP circulate in the plasma at a concentration of 23.8 mg/l. The origin of those protein bands which appear typically upon electrophoresis was still under dispute because they could not been demon- strated in proximal tubular fluid. The present study confirms the extrarenal source of SDP and suggests identity with e2u- globulin. The attempt to track down SDP from plasma to excreted urine demonstrated that, in contrast to proximal fluid, samples from nephron parts distal to the loop of Henle contain large amounts of SDP. An isolated kidney model was used to determine the sieving coefficient and tubular reabsorption of SDP, obtained from male rat urine. We have found a correlation between the sieving coefficient and the molecular weight of SDP. The sieving coefficient ranged from 0.375 to 0.834. The tubular reabsorption which has been determined with an isolated kidney perfused with albu- min and erythrocytes also showed variation with regard to molecular weight and was 61.7%, on average. Key words: Sex dependent proteinuria - Micro-disc electrophoresis - Micropuncture technique - Isolated per- fused rat kidney Introduction The rat is frequently used as a laboratory animal to study renal protein excretion although it demonstrates some diver- gence from other animal species (Alt et al. 1985). In contrast to female adult rats, male adult rats excrete large amounts of specific low molecular weight sex dependent proteins (SDP). Several authors detected sex dependent proteins by immunological methods and named them c~2u-globulin (Fin- layson et al. 1965; Neuhaus 1986). The heterogeneity of this protein could be demonstrated (Geertzen et al. 1973) consisting of four closely related proteins in polyacrylamide gel electrophoresis. This protein has been named major uri- nary protein (MUP). Offprint requests to: E. Jonas, Department of Clinical Biochemistry, Medical School Hannover, Konstanty-Gutschow-Strasse 8, D-3000 Hannover 61, Federal Republic of Germany Electrophoretic separation by polyacrylamide gels re- sulted in at least eight well defined bands (Alt et al. 1980). Since the identity of c~2u-globulin, MUP and those sex de- pendent proteins demonstrated by micro-disc electropho- resis was not clear, in the following text the more general term SDP (sex dependent proteins) will be used for all of those urinary proteins specific for male rats which are de- tected by micro-disc electrophoresis. Most molecular weight plasma proteins are almost com- pletely reabsorbed by the tubulus system after glomerular filtration (Alt et al. 1983). Preliminary micropuncture stud- ies demonstrated, however, that SDP in proximal tubular fluid were below detection limit (Jonas et al. 1984), while in distal tubular fluid large amounts of SDP were detected. These findings could be interpreted either as a selectively low uptake of filtered SDP at the tubular site or as a secretory mechanism, e.g. of the distal tubule, Henle's loop or collecting ducts. The aim of the present study was to test the hypothesis of filtration and reabsorption of SDP and to test whether or not the different proteins, as shown by disc electrophoresis, are handled alike by the kidney. Micropunc- ture techniques were used to elucidate the origin of SDP at the single nephron level in vivo. In vitro studies with isolated rat kidney were performed to investigate the renal handling of SDP. To determine the sieving coefficient of SDP, an isolated kidney fixed with glutaraldehyde was used. Using this model, the sieving coef- ficient could be determined at very low SDP concentrations because the "urine" represented unchanged ultrafiltrate which could be handled much more easily and reliably than nanoliter samples from the proximal tubule. The technique of perfusing isolated rat kidneys (Weiss et al. 1959) has been elaborated in our laboratory (Schurek 1980). This model appeared suitable for studying the renal handling of proteins at different concentrations and for testing whether or not the nature and compositions of the proteins are modified by the kidney. Moreover, the model of the isolated perfused kidney eliminated the possibility of contamination by hor- mones such as testosterone which might influence the renal handling of sex dependent proteins. Materials and methods Experimental animals. Experiments were performed with male adult Wistar crypt/Ztm rats which demonstrated uri- nary SDP patterns similar to other strains (Alt et al. 1985). The body weight of each was between 200 and 300 g. The animals had free access to food (standardized pellets,